Gram‐Scale mRNA Production Using a 250‐mL Single‐Use Bioreactor

Skok, Janja; Megušar, Polona; Vodopivec, Tina; Pregeljc, Domen; Mencin, Nina; Korenč, Matevž; Krušič, Andreja; Celjar, Anže Martinčič; Pavlin, Nejc; Krušič, Jana; Müeller, Matthias; McHugh, Kevin J.; Štrancar, Aleš; Sekirnik, Rok

doi:10.1002/cite.202200133

Cited by 20 publications

(21 citation statements)

References 27 publications

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“…mRNA for protein replacement therapies can nowadays be produced in cell-free systems through the in vitro transcription reaction (IVT). The IVT takes place in a batch or continuous bioreactor that monitors and controls reaction conditions such as pH and temperature [ 38 , 39 ]. The main IVT reagents are the linearized DNA plasmid that plays the role of the template from which mRNA is going to be transcribed, a DNA-dependent RNA polymerase that performs the transcription, dNTPs that will serve as mRNA building blocks and in some cases a 5′ Cap analogue [ 40 ].…”

Section: Considerations On Mrna Production For Protein Replacement Th...mentioning

confidence: 99%

mRNA in the Context of Protein Replacement Therapy

et al. 2023

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Protein replacement therapy is an umbrella term used for medical treatments that aim to substitute or replenish specific protein deficiencies that result either from the protein being absent or non-functional due to mutations in affected patients. Traditionally, such an approach requires a well characterized but arduous and expensive protein production procedure that employs in vitro expression and translation of the pharmaceutical protein in host cells, followed by extensive purification steps. In the wake of the SARS-CoV-2 pandemic, mRNA-based pharmaceuticals were recruited to achieve rapid in vivo production of antigens, proving that the in vivo translation of exogenously administered mRNA is nowadays a viable therapeutic option. In addition, the urgency of the situation and worldwide demand for mRNA-based medicine has led to an evolution in relevant technologies, such as in vitro transcription and nanolipid carriers. In this review, we present preclinical and clinical applications of mRNA as a tool for protein replacement therapy, alongside with information pertaining to the manufacture of modified mRNA through in vitro transcription, carriers employed for its intracellular delivery and critical quality attributes pertaining to the finished product.

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Section: Considerations On Mrna Production For Protein Replacement Th...mentioning

confidence: 99%

mRNA in the Context of Protein Replacement Therapy

et al. 2023

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“…Purity and/or isoform composition requirements vary depending on the application of the pDNA. High purity of SC pDNA isoform is required in gene therapy platforms, whereas isoform composition is usually not defined for the preparation of IVT mRNA production, where linearization step follows the pDNA purification [5][6][7]. In any case, pDNA must be purified from all other contaminants present in the E. coli lysate, such as cell debris, RNA, residual proteins and genomic DNA.…”

Section: Introductionmentioning

confidence: 99%

Analytical separation of plasmid DNA isoforms using anion exchanging chromatographic monoliths with 6 µm channels

Pavlin,

Černigoj,

Bavčar

et al. 2023

Electrophoresis

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High‐performance liquid chromatography (HPLC)‐based analytical assays are used to effectively monitor purity and quantity of plasmid DNA (pDNA) throughout the purification process. However, the phenomenon of physical entrapment of open circular (OC) isoforms pDNA inside narrow channels of chromatographic support decreases its accuracy and precision and the effect increases with pDNA size. The purpose of the study was to develop a chromatographic method for accurate analytical separation between isoforms of <16 kbp pDNA using weak anion exchanging monolithic column with large (6 µm) convective channels. Purified samples of 4.7 and 15.4 kbp large pDNA with known isoform composition were prepared and their isoforms separated in ascending salt gradient. Both OC and supercoiled (SC) isoforms were baseline separated at a flow rate below 0.5 mL min−1 in a guanidinium chloride (GdnCl) gradient with a ≥95% OC pDNA elution recovery. However, these chromatographic conditions increased 2 times the peak width for linear (LIN) pDNA isoform compared to the results using monoliths with 1.4 µm channel size. If other chaotropic agents, such as urea or thiocyanate (SCN), were added to Gdn ions, the elution volume for LIN isoform decreased. Optimization of combined GdnCl/GdnSCN gradient for pDNA elution resulted in a simple and robust chromatographic method, where OC–LIN and LIN–SC pDNA (up to 15 kbp size) were separated with resolution above 1.0 and above 2.0, respectively. The accessibility and general acceptance of anion exchange chromatography for pDNA analytics give the newly developed method a great potential for in‐process control monitoring of pDNA production processes.

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“…Plasmid DNA (pDNA) is the central molecule of modern biotechnology, both in gene therapy and vaccination, and many clinical studies for DNA vaccines against the SARS‐CoV‐2 virus are currently underway [1]. pDNA serves as the basis for the transcription of messenger RNA, which represents the active ingredient in the new generation of vaccines [2–4]. It also serves as a cell line transfection vector used in the production of adeno‐associated viruses for gene therapy [5].…”

Section: Introductionmentioning

confidence: 99%

Effect of plasmid DNA isoforms on preparative anion exchange chromatography

Kralj,

Kodermac,

Bergoč

et al. 2023

Electrophoresis

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Increased need for plasmid DNA (pDNA) with sizes above 10 kbp (large pDNA) in gene therapy and vaccination brings the need for its large‐scale production with high purity. Chromatographic purification of large pDNA is often challenging due to low process yields and column clogging, especially using anion‐exchanging columns. The goal of our investigation was to evaluate the mass balance and pDNA isoform composition at column outlet for plasmids of different sizes in combination with weak anion exchange (AEX) monolith columns of varying channel size (2, 3 and 6 µm channel size). We have proven that open circular pDNA (OC pDNA) isoform is an important driver of reduced chromatographic performance in AEX chromatography. The main reason for the behaviour is the entrapment of OC pDNA in chromatographic supports with smaller channel sizes. Entrapment of individual isoforms was characterised for porous beads and convective monolithic columns. Convective entrapment of OC pDNA isoform was confirmed on both types of stationary phases. Porous beads in addition showed a reduced recovery of supercoiled pDNA (on an 11.6 kbp plasmid) caused by diffusional entrapment within the porous structure. Use of convective AEX monoliths or membranes with channel diameter >3.5 µm has been shown to increase yields and prevent irreversible pressure build‐up and column clogging during purification of plasmids at least up to 16 kbp in size.

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Gram‐Scale mRNA Production Using a 250‐mL Single‐Use Bioreactor

Cited by 20 publications

References 27 publications

mRNA in the Context of Protein Replacement Therapy

mRNA in the Context of Protein Replacement Therapy

Analytical separation of plasmid DNA isoforms using anion exchanging chromatographic monoliths with 6 µm channels

Effect of plasmid DNA isoforms on preparative anion exchange chromatography

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