Optimizing in vitro transcription (IVT) is critical for cost-effective mRNA production. This protocol details an analytical chromatographic method for at-line analysis of IVT reaction, monitoring NTP depletion and mRNA production in batch or fed-batch modes, applicable to various RNA modalities, enhancing productivity and reducing costs.
In vitro transcription reaction (IVT) is a complex, multi-component enzymatic synthesis of mRNA from a linear DNA template, catalyzed by an RNA polymerase, e.g. T7. Due to the high cost of IVT reagents, IVT is a critical step in the mRNA drug substance production process and has been the focus of intense optimization in the field. To decrease the cost of mRNA production, reagents must be utilized optimally. Effective optimization necessitates a comprehensive understanding of the impact of individual reagents on reaction kinetics, i.e., the consumption of nucleoside triphosphates (NTPs) and the production of mRNA. Traditionally, low-throughput, end-point analytical techniques have been used for the analysis of mRNA. Though such methods give valuable information on mRNA content, at-line, near real-time analytics is needed to fully understand IVT reaction. We demonstrate how a liquid chromatography analytical method that separates NTPs, as well as pDNA and mRNA, can be used in near real-time to study IVT reaction kinetics. With the knowledge of the influence of different IVT components on kinetics and yield, rapid chromatographic analysis can be used to convert batch IVT reaction into a fed-batch mode that further increases productivity and reduces the overall cost of the reaction.
The COVID-19 pandemic has catalyzed an unprecedented revolution in biomedicine, leading to the rapid development and authorization of mRNA-based vaccines, such as BioNTech/Pfizer's Comirnaty and Moderna's Spikevax, in both Europe and the USA1,2,3. The impressive efficacy and rapid development of these vaccines have highlighted the immense therapeutic potential of mRNA technology, not only for infectious diseases but also for cancer immunotherapies, protein replacement therapies, regenerative medicine, and cellular reprogramming4,5. This era, often referred to as the mRNA revolution, underscores the need for efficient and cost-effective mRNA production processes.
The production of mRNA involves multiple unit operations, with the in vitro transcription (IVT) reaction being a critical and most cost-intensive unit operation6,7,8. The IVT reaction synthesizes mRNA from a DNA template using RNA polymerase, typically T7 RNA polymerase, and nucleoside triphosphates (NTPs) in the presence of magnesium ions. The process is relatively simple and allows for the production of large amounts of mRNA in a short time frame, achieving reaction yields of 2-5 g/L within a few hours and up to 14 g/L in some reports9,10,11. However, optimization of IVT reaction yield is crucial for reducing production costs and ensuring the scalability of mRNA vaccines and therapeutics12 and, due to sequence differences that influence NTP consumption, may be required for each construct or construct family.
The reaction is usually performed as a batch process, but recent advances in fed-batch processing and at-line analytics have opened new avenues for optimizing mRNA production7,13. Fed-batch reactions, which involve a bolus or continuous addition of reagents, can potentially extend reaction times and increase yields by preventing substrate inhibition and co-factor-dependent product degradation14,15.
The development of fast at-line analytics has been a significant advance in the field of IVT, enabling the monitoring of key reaction components such as NTPs and mRNA with minimal analytical lag13,16, adding an additional dimension to IVT monitoring, which is typically only focused on mRNA concentration17. Traditionally, mRNAs have been analyzed using techniques such as polyacrylamide gel electrophoresis (PAGE), agarose gel electrophoresis, or capillary electrophoresis. These are end-point methods and, therefore, cannot be used for real-time IVT monitoring. An alternative to analytics described in this protocol are methods using light-up RNA aptamer and fluorescence dye pairs. In this method, RNA aptamer is tagged to RNA and incubated with a light-up fluorescence dye. Transcription activity can then be visualized by fluorescence intensity17. This enables real-time analysis of the quantity and quality of transcribed mRNA during IVT 21 but does not allow simultaneous monitoring of other IVT components, e.g., NTPs. Another alternative that potentially affords real-time quantification of NTP and mRNA is Raman spectroscopy18, but to date, no report has shown its usefulness for IVT monitoring, suggesting that further optimization of the method is still required to derive the necessary selectivity and sensitivity.
Continuous development of faster and more selective analytics supports the optimization of IVT for higher yield in batch and fed-batch modes. The reaction remains a complex, multi-parameter process with multiple interacting factors, for which optima may differ between construct types (e.g., mRNA vs saRNA). The concentration and ratio of Mg2+ ions and NTPs are particularly influential, and their optimal levels must be carefully balanced to maximize mRNA yield while minimizing the formation of double-stranded RNA (dsRNA), a potent stimulant of the innate immune system19,20. Recently, feeding UTP at steady-state levels was reported as an approach to reduce dsRNA formation21. Monitoring UTP levels in near-real time would add an additional level of process control.
In this protocol, we demonstrate how at-line monitoring of the IVT reaction can increase the yield of mRNA production in batch and fed-batch mode.
1. Buffer preparation
NOTE: All buffers must be prepared RNase-free, meaning that all chemicals and glassware must be used only for RNase-free work and handled with precaution. Water used for buffer preparation and cleaning of glassware must be certified as nuclease-free. Before conducting an experiment, all work surfaces and glassware must be sprayed with a decontamination reagent that eliminates RNases. The decontamination reagent must be thoroughly washed away with RNase-free water before glassware/work area is used. If possible, use sterile and one-use-only consumables.
2. Preparation of in vitro transcription reaction (IVT)
NOTE 1: IVT reaction can be a batch reaction, where all reagents are added at the beginning, and the reaction is stopped after NTPs are depleted/mRNA production reaches a plateau, or can be a fed-batch reaction, where depleted NTPs are replenished with additional NTPs and with that mRNA production additionally increases. Linear pDNA template used in IVT reaction can be obtained by using restriction enzyme that cleaves right after poly(A) sequence in plasmid DNA or by PCR reaction. In both cases the reaction must be followed by purification, either chromatographic or using commercial DNA purification kits.
3. Preparation of chromatographic analysis
4. Sample quantification and data analysis
The chromatographic analysis described in this protocol can be used either for IVT optimization or for converting a batch IVT reaction into a fed-batch reaction (Figure 1).
To test the effect of different buffer compositions on the kinetics of IVT reaction, three different IVT reactions were mixed according to the protocol written in Table 2. Buffer containing Tris, where pH was adjusted with HCl, was compared to Tris buffer, where pH was adjusted with acetic acid. Further, both Tris buffers were compared with the HEPES buffer, where pH was adjusted with NaOH. All 1x IVT buffer's composition was 40 mM Tris/HEPES, 10 mM DTT, 2 mM spermidine, and pH 7.9.
From 100 µL of IVT reactions mix, 2 µL of IVT sample was taken out of the IVT reaction and quenched with 2 µL of 100 mM EDTA every 15 min in the first hour of incubation and then every 30 min until 180 min of incubation. The expected final mRNA yield for this reaction condition was 15 mg/mL, meaning that 2-fold dilution due to quenching was followed by 400-fold dilution in MPA+NaCl for analysis. The sample was diluted 800-fold in total, meaning that even at 15 mg/mL production, mRNA concentration loaded onto the analytical chromatographic column was still within calibration curve concentrations.
NTP and mRNA areas at A260 were integrated for each individual sample at each individual timepoint (tx). Areas were then converted into concentrations for mRNA, using the mRNA calibration curve and consumption percentage for NTPs, using A260 area of NTPs at 0 min (t0) as a 100% (see equation below).
Results can be shown as graphs for each individual IVT reaction, where time is shown on the x-axis and mRNA concentration and remaining NTPs are shown on the y-axis (Figure 2A). All IVT, meaning mRNA concentrations in dependence on time, can also be plotted together in one graph, and mRNA production kinetics can be studied and optimal IVT conditions selected (Figure 2B).
For the fed-batch experiment, IVT was mixed as written in Table 3. From 300 µL of IVT reaction mix, 2 µL of IVT sample was removed from the IVT reaction and quenched with 2 µL of 100 mM EDTA every 30 min. Additionally, the sample was taken immediately after each bulk NTP+MgCl2 addition. A feed solution containing 42.4 mM of each NTP and 152.5 mM MgCl2 was prepared in advance, and 106 µL of each individual 200 mM NTP was mixed with 76.2 µL of 1 M MgCl2. A feeding regime was established, where bolus feed was added every hour (at 60 min, 120 min, 180 min, and 240 min of incubation). The feeding regime is described in Table 3 Samples were analyzed in near real-time with analytics as described for bulk IVT reaction. After 300 min of monitoring the IVT reaction was quenched.
Results can be shown as remaining NTPs/mRNA production over time of incubation. Since bolus feed additions also dilutes IVT reaction, mRNA concentration in IVT drops at each feed addition (Figure 3A). Increase in mRNA mass can also be measured and presented by transcription factors (defined as mmRNA/mpDNA), showing linear increase in mRNA production over time (Figure 3B).
Figure 1: Schematic overview (A) Representation of IVT optimization workflow with at-line chromatographic analysis quantification of NTPs and mRNA. (B) Representative chromatograms of a batch IVT reaction sampled at (i) t0, (ii) mid-point, (iii) final reaction time-points. Please click here to view a larger version of this figure.
Figure 2: Batch IVT graphs. (A) Representative graph of IVT reaction with mRNA production and NTP consumption on the y-axis. mRNA concentration plateau visible at 120 - 180 min of incubation which correlates with NTP consumption as limiting NTP (ATP) is consumed at 120 min. (B) IVT graph showing the influence of IVT buffer on IVT kinetics. IVT containing Buffer A (Tris+acetic acid) showed the fastest mRNA production, followed by Buffer B (Tris+HCl) and Buffer C (HEPES+NaOH) as the slowest. Please click here to view a larger version of this figure.
Figure 3: Fed-batch IVT graph. (A) IVT graph where NTP+MgCl2 feeds were added at 60-, 120-, 180-, and 240-min. mRNA concentration in the IVT reaction drops at every feed addition due to dilution with NTP+MgCl2 feed. (B) Increase in mRNA mass showed by transcription factor is linear throughout the reaction incubation. Please click here to view a larger version of this figure.
Table 1: Generic IVT protocol. Please click here to download this Table.
Table 2: Batch IVT protocol. Please click here to download this Table.
Table 3: Fed-batch IVT protocol. Please click here to download this Table.
In this method, IVT samples are analyzed at different time points during incubation using an analytical chromatographic multimodal column that separates NTPs, pDNA, and mRNA, thus allowing close monitoring of NTP consumption and mRNA production. This method measures NTPs and mRNA concentration in a quantitative manner based on changes in A260 areas at designated time points. Since the method provides information on NTP and mRNA concentration, it is highly suitable for IVT optimization, where the primary goal is often to maximize mRNA yield and minimize reaction time; therefore, understanding the effect of different IVT reagents on the kinetics of mRNA production is critical22. We show how at-line monitoring can be applied to optimize IVT reaction in batch and fed-batch modes.
The main advantage of this method is an at-line analysis that allows for near-real-time monitoring of IVT as each sample is analyzed in sub-8 min. Preparation of samples for analytics is straightforward, as only dilution in MPA is required without any prior sample pre-treatment. The sample volume required for analysis is very low, e.g., 1 µL of IVT. This small pipetting volume could potentially result in analytical deviations due to pipetting errors. However, as the high throughput of the analytical approach allows for triplicate measurements, outliers are easily identifiable, and deviations from the expected kinetic curve are not hard to detect.
One of the limitations of the method is the chromatographic co-elution of UTP and CTP. Precise quantification of UTP and CTP is usually not necessary as the limiting NTP in an mRNA sequence is often either ATP or GTP. If separate quantification of UTP and CTP is required, the difference in UV absorbance at 260 nm and 280 nm for UTP and CTP can be harnessed to derive the relative abundance of each NTP in the chromatographic peak.
This analytical method does not separate RNA species above 100 nucleotides in length; therefore, it cannot be used to detect differences in mRNA quality, e.g., it does not differentiate between non-polyadenylated and polyadenylated mRNA, between dsRNA or abortive transcripts and ssRNA and is not suitable for stability studies as it cannot differentiate between degraded and nondegraded mRNA. However, the method can be utilized for the quantification of other RNA modalities, such as circRNA, tRNA, and saRNA. Although these molecules vary in size and structure, the same analytical method can be employed to study the production yields of each in an IVT reaction.
The authors have nothing to disclose.
The authors would like to thank Tomas Kostelec, Blaž Bakalar, Nejc Pavlin, Andreja Gramc Livk, and Anže Martinčič Celjar for helpful discussions.
Name | Company | Catalog Number | Comments |
0.22 µm PES membrane filter, e. g. Sartolab BT 500 | Sartorius | 180E14---------E | |
0.5 mL plastic tubes; e. g. e. g. DNA LoBind, PCR clean | Eppendorf | 30108035 | |
1.5 mL plastic tubes; e. g. DNA LoBind, PCR clean | Eppendorf | 30108051 | |
ATP Solution | MEBEP BIOSCIENCE | R1331T2 | |
Benchtop cooler -20 °C | Brand | 114935 | |
CIMac PrimaS 0.1 mL Analytical Column (2 µm) | Sartorius BIA Separations | 110.5118-2, 2 µm channels | |
CTP Solution | MEBEP BIOSCIENCE | R3331T2 | |
DTT | Sigma | 10197777001 | |
EDTA-Na2 x 2H2O | Kemika | e.g. 11368 08 | |
GTP Solution | MEBEP BIOSCIENCE | R2331T2 | |
HEPES | Merck | 1.10110.1000 | |
HPLC high recovery vial | Macherey-Nagel | 702860 | |
MgCl2 | Invitrogen | AM 9530G | |
Microvolume spectrophotometer | Thermo Scientic | Nanodrop | |
mRNA standard, mFix4, 4000 nt | Sartorius BIA Separations | BIA-mFix4.1.1 | |
Na4P2O7 + 10 H2O | Sigma | S6422-500G | |
NaCl | Fluka | 31434-1KG-M | |
NaOH | Merck | 1064691000 | |
PATfix mRNA analytical platform | Sartorius BIA Separations | PAT0021 | |
Pipette 100 – 1000 μL | Eppendorf | Reference 2 | |
Pipette 2 – 10 μL | Eppendorf | Reference 2 | |
Pipette 20 – 200 μL | Eppendorf | Reference 2 | |
Pyrophosphatase | MEBEP BIOSCIENCE | M2403L | |
Rnase Away Decontamination Reagent | Thermo Scientic | 10328011 | |
RNAse inhibitor | MEBEP BIOSCIENCE | RNK3501 | |
Spermidine | Sigma | 85558-5G | |
T7 mRNA polymerase | MEBEP BIOSCIENCE | TR01 | |
Thermoblock | Thermo Scientic | EPPE5382000.015 | |
Trizma Base | Sigma | T6066-1KG | |
UTP Solution | MEBEP BIOSCIENCE | R5331T2 | |
Vial caps | Macherey-Nagel | 70245 | |
Vortex | IKA | V1900 |
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