mRNA production using the ReadyToProcess WAVE™ 25 Rocker

The COVID-19 pandemic and subsequent emergence of messenger RNA (mRNA) vaccines has revolutionized RNA-based genomic medicine driving an explosion of research and clinical interest (1-3). The American Society for Gene and Cell Therapy reports 29 approved RNA therapies with a further 1016 in the clinical pipeline as of Q4 2023. Applications for these therapies include rare disease treatment, infectious disease prophylaxis, and cancer therapy among other indications (1-3). The intensification of interest in mRNA has spurred the demand for mRNA manufacturing capabilities. We will show that the ReadyToProcess WAVE™ 25 Rocker system offers a complete platform for the GMP linearization of template DNA and subsequent production of mRNA.

Introduction

The production process of mRNA therapeutics consists of: (1) drug discovery/design, (2) DNA template preparation and purification, (3) mRNA synthesis, (4) mRNA purification, (5) drug formulation, and (6) fill-finish (Fig 1). Bacterial fermentation is currently the workhorse of the industry for plasmid DNA (pDNA) production and is followed by restriction endonuclease treatment to yield a linear DNA template (2). mRNA synthesis is typically performed by in vitro transcription (IVT), a cell-free enzymatic reaction where RNA polymerase catalyzes the synthesis of RNA from nucleotide triphosphates (NTPs) using the linear DNA template. Although a relatively straightforward reaction, IVT nevertheless requires strict control to inhibit generation of immunostimulatory by-products, reduce the risk of nuclease contamination and adhere to evolving regulatory requirements (2).

Fig 1. Overview of the RNA manufacturing process.

Materials and methods

Linearization

Linearization of plasmid DNA requires a restriction endonuclease and an appropriate salt containing buffer which is generally optimized for the enzyme. The DNA construct used in this example process encoded enhanced green fluorescent protein (eGFP) and contained an EcoRI restriction site downstream of the gene of interest. A commercially available EcoRI was used in conjunction with a 10× proprietary buffer solution provided by the enzyme vendor. Small scale linearization reactions were performed in Eppendorf tubes placed in an incubator at 37°C which is the optimal temperature for EcoRI. Once appropriate DNA and enzyme concentrations were determined, large-scale linearizations were performed in the ReadyToProcess WAVE™ 25 Rocker using a 2 L Cellbag™. At this scale the reaction mixture was allowed to pre-heat to the setpoint temperature of 37°C prior to addition of EcoRI and initiation of the reaction. Prior to use in any IVT reaction, linearized DNA template was purified via tangential flow filtration (TFF) with a 100 MWCO hollow-fibre membrane followed by 0.2 µm filtration.

IVT

In general, IVT requires RNA polymerase, NTPs, the linear DNA template, a source of Mg2+ ions as a cofactor, and a reaction buffer. RNase inhibitor and inorganic pyrophosphatase (IPP) can also be included to mitigate against RNase degradation of the mRNA product and inhibit the formation of magnesium pyrophosphate respectively. Optimization of the IVT recipe is a topic of much study but with limited consensus among practitioners (1, 2, 4, 5). The recipe used in this example process was chosen to ensure a yield ≥ 4 g/L of the eGFP mRNA and, unless otherwise mentioned, is laid out in Table 1. Because of limited pDNA supply a concentration of 50 µg/mL was used during initial scale-up studies while 150 µg/mL was used in the 1 L and 2 L runs to maximize mRNA yield.

IVT reactions were performed at small scale (0.1 to 5 mL) in Eppendorf or conical tubes placed in a shaking incubator at 37°C and 300 rpm for 2 h. For scales between 120 mL to 2 L, the IVT reactions were performed on the ReadyToProcess WAVE™ 25 Rocker in 2 L or 10 L Basic Cellbag™ which were chosen for simplicity as they included the minimum required functionality. At larger scales the reaction mixture was allowed to pre-heat to the setpoint temperature of 37°C prior to addition of T7 RNA polymerase and initiation of the reaction. After 2 h, products were treated with 3.5 µg/mL DNAse and 2.5 mM CaCl2 for 40 min to digest the DNA template. The reaction was then quenched by addition of EDTA to a final concentration of 80 mM and harvested from the Cellbag™ which were then rinsed by two successive additions of 40 mM Tris, using volumes equivalent to the final IVT reaction volume (i.e., 1.16 L or 2.32 L). This resulted in a 3× dilution of the IVT solution.

Table 1. IVT reaction components

*enzymes were sourced from Aldevron LLC

Analytics

Analysis of the linearization was performed using gel electrophoresis. Circular DNA constructs produce two distinct bands representing the open circular and super coiled structures, whereas, a completely linearized product should produce a single band at a size of 6000 nucleotides (nt).

Unless otherwise specified all analytics from the development IVTs were performed on samples that were purified using LiCl precipitation. The mRNA concentration was measured spectrophotometrically; percent integrity was measured by capillary electrophoresis (Agilent TapeStation) and is defined as the ratio of the main peak area to the total area; and, double stranded RNA (dsRNA) content was measured by J2 immunoblot assay using a 142 basepair (bp) control dsRNA.

Unpurified samples from the 1 L and 2 L production runs were used to assess mRNA concentration (Ribogreen fluorescence assay), percent integrity (SciEx PA800 Plus capillary electrophoresis system), residual DNA content (resDNASEQ kanamycin resistance qPCR kit), residual protein content (NanoOrangeTM fluorescent quantitation assay), and RNase contamination (RNaseAlertTM fluorescence assay). dsRNA content was measured as described above on a LiCl purified sample.

To determine protein expression (potency), purified mRNA samples were post-transcriptionally capped with vaccinia capping enzyme and 2’-O-methyltransferase, purified a second time with LiCl precipitation and then transfected to BHK 570 cells using jetMESSENGER transfection reagent; the percentage GFP positivity was determined 24 h after transfection using cell-based high throughput fluorescence microscopy using the Cytation 7 platform or flow cytometry using the Cytoflex.

Results and discussion

Linearization process development

Small scale linearization reactions were first performed at 50 µL to determine effective enzyme loading. As shown in Figure 2, complete linearization was observed after 1 h for all enzyme concentrations tested, with no non-specific activity as evidenced by the single band at 6000 nt. A reaction with 1 U/µg was then scaled up almost 1000-fold to 40 mg of DNA. Again, complete digestion was observed after 1 h (Fig 3). Transfer to the ReadyToProcess WAVE™ 25 Rocker was then performed as the reaction was further scaled up to 120 mg and 200 mg (Fig 4). Enzyme concentration was maintained at 1 U/µg DNA in these reactions although DNA concentration varied from 0.35 to 0.9 mg/mL to modify total volume. Samples taken every 15 min showed incomplete digestion for the 0.9 mg/mL run up to 30 min, although in both cases full digestion was achieved by 1 h (Fig 4).

Fig 2. 1% agarose gel images for 50 µL DNA linearization reactions performed at 37°C and 1 h for different EcoRI enzyme loading. Non-linearized plasmid DNA was run as a control in the lane marked “circDNA” and the two bands represent open circular and supercoiled forms.

Fig 3. 1% agarose gel images for DNA linearization reactions of different scales performed with an EcoRI concentration of 1 U/µg DNA at 37°C and 1 h. Non-linearized plasmid DNA was run as a control in the lane marked “circDNA” and the two bands represent open circular and supercoiled forms.

Fig 4. 1% agarose gel images for the linearization of (A) 120 mg of DNA at 0.35 mg/mL, and (B) 200 mg of DNA at 0.9 mg/mL. Reactions performed on the ReadyToProcess WAVE™ 25 Rocker with EcoRI concentration of 1 U/µg DNA at 37°C and 1 h. Non-linearized plasmid DNA was run as a control in the lanes marked “circDNA” and the two bands represent open circular and supercoiled forms.

IVT Scale Up

IVT yield for a 1 kb mRNA construct was found to be scale independent from 1.5 mL to 1000 mL with the total amount of mRNA produced directly proportional to the reaction volume (Fig 5). Reaction rate was also found to be consistent across the reaction scales with mRNA concentration increasing linearly out to 2 h (Fig 6). The linear increase in mRNA yield suggested that the reaction does not become reagent limited over 2 h and that additional mRNA could be produced from the same amount of NTPs by extending the duration of the reaction or by increasing the reaction rate through addition of more DNA template and/or T7 enzyme.

Reaction scale was also found to have no impact on the mRNA quality. The dsRNA content and mRNA integrity remained consistent at all scales and the protein expression from mRNA produced at large scale on the ReadyToProcess WAVE™ 25 Rocker and at small scale in an Eppendorf tube showed no difference (Figs 7 and 8).

Fig 5. (A) mRNA yield and (B) total mass produced from IVT reactions performed at different scales in tubes (1.5 mL to 5 mL) or on the ReadyToProcess WAVE™ 25 Rocker (≥ 120 mL). Data points are average of three measurement replicates of a single reaction sample.

Fig 6. Yield of mRNA over time for IVT reactions performed at different scales in a tube (1.5 mL) or on the ReadyToProcess WAVE™ 25 Rocker (≥ 120 mL). Data points are mean of three measurement replicates of a single reaction sample.

Fig 7. (A) dsRNA content and (B) integrity of mRNA produced from IVTs of different scales in tubes (1.5 mL to 5 mL) or on the ReadyToProcess WAVE™ 25 Rocker (≥ 120 mL). (C) Electropherograms from capillary electrophoresis (CE) analysis of each sample and (D) example % integrity calculation for the 1000 mL sample, where % integrity is the ratio of the peak between 810 to 1400 bp to the total peak area. Data points in (A) are mean of three measurement replicates of a single sample. Data points in (B) are single measurement of a single sample.

Fig 8.GFP expression 24 h post transfection in BHK 570 cells of mRNA produced from IVTs performed at small-scale and on the ReadyToProcess WAVE™ 25 Rocker.

Use of the ReadyToProcess WAVE™ 25 Rocker enabled the capture of process data throughout the IVT reaction. As shown in Figure 9, pre-heating of the reaction solution took approximately 40 min at both 120 mL and 1 L scales. Reaction volume did, however, impact the temperature drop during T7 addition and subsequent reaction sampling. Temperature in the 120 mL reaction dropped by 3.5°C during T7 addition and 1.1°C at each sampling point as compared to associated drops of only 0.3°C and 0.1°C at 1L. A similar phenomenon occurred during DNase addition and may have to be accounted for during process development to ensure optimal performance of the enzymes.

Fig 9. ReadyToProcess WAVE™ 25 Rocker data from (A)120 mL and (B) 1 L IVT reactions. (a) addition of T7 and reaction initiation; (b) sampling points; (c) addition of DNase and CaCl2; (d) reaction quench with EDTA; (e) harvest.

Although not used in this study, Cellbag™ biocontainers can include a probe for in-line monitoring of pH. In order to evaluate the utility of this functionality a 150 mL IVT reaction was performed for 4 h on the ReadyToProcess WAVE™ 25 Rocker with samples taken every 30 min for mRNA content and off-line pH analysis. Considering the reaction chemistry wherein an H+ ion is released for every nucleotide addition to mRNA it was expected to see decreasing pH with increasing mRNA production and that was indeed found to be the case (Fig 10). In fact, the observed linear relationship between mRNA and pH (Fig 11), if verified for specific constructs and reaction conditions, could act as a real-time, sample free, proxy metric for the progression of the IVT reaction as has been previously proposed (6). This would be an additional benefit of performing IVTs in the ReadyToProcess WAVE™ 25 Rocker system.

Fig 10. ReadyToProcess WAVE™ 25 Rocker data from (A)120 mL and (B) 1 L IVT reactions. (a) addition of T7 and reaction initiation; (b) sampling points; (c) addition of DNase and CaCl2; (d) reaction quench with EDTA; (e) harvest.

Fig 11. mRNA yield as a function of solution pH (measured off-line) from a 150 mL IVT reaction performed on the ReadyToProcess WAVE™ 25 Rocker.

1 L to 2 L IVT using ReadyToProcess WAVE™ 25 Rocker

Following process development work an end-to-end production run was executed to produce approximately 15 g of mRNA product for further downstream processing and encapsulation studies. As part of that workflow, a DNA linearization was performed in ReadyToProcess WAVE™ 25 Rocker systems to produce 920 mg of linear DNA template. The conditions established in the development work were used for this reaction, EcoRI loading at 1 U/µg DNA and 0.9 mg/mL DNA.

Prior to initiation of the enzymatic digestion, the reaction solution was pre-heated to 37°C  which took approximately 40 min (Fig 12). Addition of the EcoRI saw a decrease in temperature of 2.8°C which re-stabilized within 15 min. Sampling time points had less of an impact on temperature, decreasing it by only 0.5°C. Time course testing showed complete linearization of the DNA within 15 min of reaction initiation (Fig 13). Moreover, the process recovery was found to be 96%.

Fig 12. ReadyToProcess WAVE™ 25 Rocker process data from large-scale linearization reaction. (a) addition of EcoRI and reaction initiation; (b) sampling; (c) Cellbag™ biocontainer harvest.

Fig 13.1% agarose gel images for the linearization of 920 mg of DNA performed on the ReadyToProcess WAVE™ 25 Rocker with EcoRI concentration of 1 U/µg DNA at 37°C and 1 h. Non-linearized plasmid DNA was run as a control in the lane marked circDNA and the two bands represent open circular and supercoiled forms.

The linearized DNA template was then, after TFF purification, used in two IVT reactions performed on the ReadyToProcess WAVE™ 25 Rocker at 1 and 2 L scales.  The same IVT reaction recipe used in the development work was used for these reactions with the exception that the DNA concentration was increased to 150 µg/mL in order to maximize utilization of NTPs and mRNA yield.

Similar to the development work, pre-heating the reaction solution took approximately 40 min after which the temperature was maintained at the set-point, apart from when the T7 polymerase was added to initiate the reaction and when the DNase and CaCl2 were added after transcription to digest template DNA (Fig 14). Deviations in temperature at these points were slight (approximately 0.5°C) and took approximately 12 min to restabilize.

Fig 14. ReadyToProcess WAVE™ 25 Rocker process data from large-scale (A) 1 L and (B) 2 L IVT reactions. (a) addition of T7 and reaction initiation; (b) addition of DNase and CaCl2; (c) reaction quench with EDTA; (d) Cellbag™ biocontainer rinse and harvest.

The 1 L and 2 L production IVT runs were found to produce 7.10 and 6.92 g/L of mRNA respectively (Table 2). The increase in yield as compared to what was observed during scale-up activities was due to the increased DNA concentration that was used in the larger runs as previously discussed. In both cases dsRNA content was < 1% and no RNase activity was detected. Moreover, residual DNA content was found to be 59 ng DNA/mg RNA which would be below regulatory specifications of 10 ng/dose for typical mRNA products even prior to downstream processing (7). Potency was found to be similar to that from process development work, with 57% GFP positivity. mRNA integrity was 100% in both scales (Fig 15). This differs from the measured integrity during development work which was likely due to differences in analytical equipment/methodology rather than a difference in product quality.

Table 2. Results from large-scale IVT process on ReadyToProcess WAVE™ 25 Rocker study

*performed on a sample taken after the 1 L and 2 L reaction products were pooled together prior to downstream processing

Fig 15. Electropherograms (SciEx PA800 system) of mRNA from (A) 1 L and (B) 2 L IVT reactions performed on the ReadyToProcess WAVE™ 25 Rocker system. Analysis was performed on crude samples.

Conclusions

We have shown:

• The successful scale up of plasmid DNA linearization and IVT reaction from Eppendorf tubes to ReadyToProcess WAVE™ 25 Rocker system.
• The production of 920 mg of linear DNA template and 20 g of mRNA with excellent product yields and qualities, demonstrating the utility of the ReadyToProcess WAVE™ 25 Rocker system for GMP production of mRNA therapeutics.

References

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