Development and scale-up of oligo-dT monolithic chromatographic column for mRNA capture through understanding of base-pairing interactions

Mencin, Nina; Štepec, Dona; Margon, Alja; Vidič, Jana; Dolenc, Darko; Simčič, Tina; Rotar, Sara; Sekirnik, Rok; Štrancar, Aleš; Černigoj, Urh

doi:10.1016/j.seppur.2022.122320

Cited by 14 publications

(14 citation statements)

References 27 publications

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“…DBCs around 7 mg pDNA per mL of column were consistent with expectations and literature data [11] for the smallest, 7.3 kbp large pAAV2/8 molecule. The DBCs of columns with flow channel diameters of 3 and 6 µm were expectedly lower in accordance with the reduction of the surface area (Table 2) of the monoliths with larger channels [29, 31].…”

Section: Resultsmentioning

confidence: 83%

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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Section: Resultsmentioning

confidence: 83%

Effect of plasmid DNA isoforms on preparative anion exchange chromatography

Kralj,

Kodermac,

Bergoč

et al. 2023

Electrophoresis

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“…Porous poly dT media was reported to have a binding capacity of about 2–4 mg mRNA/mL (Mencin et al, 2023). To investigate the feasibility of in situ capping of poly dT‐tethered mRNA, the LiCl‐purified mRNA was mixed with poly dT at a binding ratio of 2 μg mRNA/μL, so that efficient mRNA adsorption would be ensured.…”

Section: Resultsmentioning

confidence: 99%

“…Since the capping reaction takes place inside the poly‐dT media, it is important to ensure highly efficient mRNA binding and enzymes entering into the pores of the media. It was reported that the poly‐dT media have an mRNA binding capacity of 2–4 mg/mL (Mencin et al, 2023), therefore, we then examined the in situ capping of poly dT‐tethered mRNA at four different mRNA/poly dT ratios of 1, 2, 3, and 4 μg‐mRNA/μL‐media. Figure 4b summarizes the results of mRNA binding/elution/capping efficiency and overall recovery.…”

Section: Resultsmentioning

confidence: 99%

On‐column capping of poly dT media‐tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase

Che,

Feng,

et al. 2023

Biotech & Bioengineering

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The messenger RNA (mRNA) 5′‐cap structure is indispensable for mRNA translation initiation and stability. Despite its importance, large‐scale production of capped mRNA through in vitro transcription (IVT) synthesis using vaccinia capping enzyme (VCE) is challenging, due to the requirement of tedious and multiple pre‐and‐post separation steps causing mRNA loss and degradation. Here in the present study, we found that the VCE together with 2′‐O‐methyltransferase can efficiently catalyze the capping of poly dT media‐tethered mRNA to produce mRNA with cap‐1 structure under an optimized condition. We have therefore designed an integrated purification and solid‐based capping protocol, which involved capturing the mRNA from the IVT system by using poly dT media through its affinity binding for 3′‐end poly‐A in mRNA, in situ capping of mRNA 5′‐end by supplying the enzymes, and subsequent eluting of the capped mRNA from the poly dT media. Using mRNA encoding the enhanced green fluorescent protein as a model system, we have demonstrated that the new strategy greatly simplified the mRNA manufacturing process and improved its overall recovery without sacrificing the capping efficiency, as compared with the conventional process, which involved at least mRNA preseparation from IVT, solution‐based capping, and post‐separation and recovering steps. Specifically, the new process accomplished a 1.76‐fold (84.21% over 47.79%) increase in mRNA overall recovery, a twofold decrease in operation time (70 vs. 140 min), and similar high capping efficiency (both close to 100%). Furthermore, the solid‐based capping process greatly improved mRNA stability, such that the integrity of the mRNA could be well kept during the capping process even in the presence of exogenously added RNase; in contrast, mRNA in the solution‐based capping process degraded almost completely. Meanwhile, we showed that such a strategy can be operated both in a batch mode and in an on‐column continuous mode. The results presented in this work demonstrated that the new on‐column capping process developed here can accomplish high capping efficiency, enhanced mRNA recovery, and improved stability against RNase; therefore, can act as a simple, efficient, and cost‐effective platform technology suitable for large‐scale production of capped mRNA.

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“…Unlike affinity chromatography where DBC does not differ between mRNA in IVT mixture and pre-purified mRNA because the ligand only binds the target molecule, lower binding capacity for mRNA from IVT with mixedmode PrimaS is likely due to weak competitive binding of charged impurities (NTPs, pDNA) to the PrimaS ligand. Further DBC increase could potentially still be achieved by adjusting binding conditions for mRNA such that all other impurities elute in flow-through, but we have not pursued further optimization as 5-7 mg/mL binding capacity is higher than average binding capacity reported for industrial affinity ligands for mRNA under standard conditions (2-4 mg/mL [19,31]).…”

Section: Dynamic Binding Capacitymentioning

confidence: 99%

“…Common mRNA purification techniques include traditional precipitation (e.g., by LiCl), tangential flow filtration (TFF) or liquid chromatography-based size exclusion, affinity, ion exchange, hydrophobic interaction and reverse-phase approaches, reviewed elsewhere [17]. The most industrially scalable chromatographic purification strategy for mRNA is affinity chromatography, utilizing the strong and selective interaction between 3′ polyadenylic acid (polyA) tail of mRNA with oligo-deoxythymidylic acid (Oligo dT) chain immobilized onto chromatographic supports [18,19]. Although highly selective, Oligo dT chromatography cannot be used for non-polyadenylated constructs, which can include a subclass of mRNA constructs requiring posttranscriptional polyadenylation, as well as circular RNA (circRNA) constructs, which are gaining attention due to increased in vivo stability compared to mRNA [20,21].…”

Section: Introductionmentioning

confidence: 99%

Scalable multimodal weak anion exchange chromatographic purification for stable mRNA drug substance

Megušar,

Miklavčič,

Korenč

et al. 2023

Electrophoresis

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Messenger RNA (mRNA) has emerged as a modality with immense therapeutic potential. Recent innovations in production process of mRNA call for procedures to isolate pure mRNA drug substance (DS) with high yield, high capacity, scalability, and compatibility with GMP production systems. Novel RNA modalities, such as circular RNA (circRNA), have further driven the need for non‐affinity capture possibilities which are already widely used in the biopharmaceutical industry, for example, in monoclonal antibody processing. The principle that multimodal ion exchange/hydrogen bonding chromatography can be used to separate mRNA from in vitro transcription components has recently been demonstrated. Here, we apply and refine this approach to be suitable for scalable purification of multiple mRNA constructs with sufficient yields, purity, and stability, for use in mRNA production process. Binding capacity of the PrimaS‐modified monolithic chromatographic column for mRNA enabled up to 7 mg/mL product isolation in a single chromatographic run, with 98% recovery and room temperature stability of the eGFP mRNA demonstrated for up to 28 days. This approach is independent of construct size or the presence of polyadenylic acid tail and is applicable for capture of a wide variety of RNAs, including mRNA, self‐amplifying RNA, circRNA, and with optimization also smaller RNAs such as transfer RNA and others.

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Development and scale-up of oligo-dT monolithic chromatographic column for mRNA capture through understanding of base-pairing interactions

Cited by 14 publications

References 27 publications

Effect of plasmid DNA isoforms on preparative anion exchange chromatography

Effect of plasmid DNA isoforms on preparative anion exchange chromatography

On‐column capping of poly dT media‐tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase

Scalable multimodal weak anion exchange chromatographic purification for stable mRNA drug substance

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