In recent years, mRNA technology has emerged as a transformative breakthrough in the biopharmaceutical industry. Characterized by rapid research and development cycles, flexible production scalability, and precise control over protein expression, mRNA therapeutics and vaccines exhibit broad application prospects in infectious disease prevention and treatment, tumor immunotherapy, and the management of hereditary diseases. Compared with traditional pharmaceuticals, mRNA can direct the human body to generate therapeutic proteins endogenously and boasts tremendous development potential.
Commercial manufacturing of mRNA predominantly relies on in vitro transcription (IVT). In this process, target mRNA molecules are synthesized using a DNA template under catalysis by specific enzymes. Nevertheless, in vitro transcription is not a faultless reaction. While generating the desired single-stranded mRNA, numerous product-related and process-related impurities are concomitantly produced, including mRNA aggregates, truncated transcriptional byproducts, and immunogenic double-stranded RNA (dsRNA). These impurities not only markedly compromise mRNA purity and production yield but also have the potential to activate the human innate immune system, trigger inflammatory adverse reactions, and even inhibit the translation and synthesis of target proteins.
Accordingly, efficient removal of such impurities to guarantee the safety and efficacy of final products constitutes a core challenge in mRNA manufacturing. Regulatory guidelines governing mRNA purity standards are being progressively refined, yet quantitative control thresholds for various impurities remain under exploration. This article briefly discusses key impurity species generated during mRNA production, their formation mechanisms, and corresponding control strategies. A comparative analysis of prevailing purification technologies and quality testing methods is also presented, aiming to provide references for process optimization and quality control of mRNA pharmaceuticals.
Double-Stranded RNA (dsRNA) and Its Process Control
During mRNA production, dsRNA represents the primary impurity generated from the IVT process. Based on structural features, dsRNA is generally categorized into two types: hairpin dsRNA formed via intramolecular folding and self-complementary base pairing of single-stranded RNA; and sense-antisense dsRNA formed by hybridization of two complementary RNA strands. The formation mechanisms of these impurities are complex, mainly attributed to off-target enzymatic activities. For instance, polymerases may undergo terminal self-extension during transcription to form hairpin structures, or initiate transcription on the non-template strand to generate antisense RNA fragments.
Widespread presence of dsRNA severely impairs the efficacy and safety of mRNA products. Specific immune sensors in the human body can recognize these characteristic structures, thereby triggering immune responses that induce interferon release and inflammatory side effects. Furthermore, such immune reactions shut down intracellular protein synthesis pathways, leading to drastically reduced expression levels of target proteins. It is therefore critical to implement multiple strategies to mitigate dsRNA formation during manufacturing.
Strategies for dsRNA reduction fall into the following categories:
Optimization of reaction substrates and components: Incorporation of modified nucleotides (e.g., 1-methylpseudouridine) or adjustment of key ion concentrations in buffers (such as reduced magnesium chloride concentration) can partially suppress dsRNA generation.
Process conditions and purification improvement: Elevated transcription temperatures or introduction of mild denaturants (e.g., urea or formamide) can disrupt undesired base pairing. In addition, high-resolution chromatographic purification of the starting DNA template significantly minimizes non-specific transcription induced by template impurities.
Enzyme engineering optimization: Genetic modification of RNA polymerases enables sustained high yields while reducing the propensity to produce short fragments and hairpin impurities.
In terms of quality control and detection, antibody-based immunoassays (e.g., ELISA) are widely adopted for rapid qualitative or semi-quantitative identification of dsRNA. For refined process development and manufacturing workflows, ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) offers greater advantages. Leveraging differences in molecular hydrophobicity, this technique achieves effective separation and quantitative analysis of target single-stranded mRNA and dsRNA impurities under high-temperature denaturing conditions, and has become a core platform for purity assessment. Alongside advances in analytical technologies, emerging high-throughput approaches such as nanopore sequencing are delivering powerful support for in-depth evaluation of mRNA integrity and sequence fidelity.
Formation and Control of mRNA Fragments
Apart from dsRNA, truncated transcriptional products (short mRNA fragments) represent another prevalent class of process impurities in mRNA manufacturing. These fragments are primarily generated at the initial stage of IVT. Upon recognition of the DNA promoter and initiation of synthesis, RNA polymerases form an unstable initiation complex, which is prone to premature transcription termination and releases short fragments ranging from 2 to 13 nucleotides in length — a phenomenon termed abortive transcription.
As transcription proceeds, once the nascent RNA chain reaches a certain length (typically exceeding 10 nucleotides), the polymerase undergoes substantial conformational rearrangement and transitions into a highly stable elongation phase. If the transition from initiation to elongation fails, repeated transcriptional abortion occurs, generating abundant non-functional short fragments. Beyond fragments originating from transcription, hydrolysis or RNase contamination in downstream workflows can cause full-length mRNA degradation and generate additional undesired RNA fragments.
Accumulation of mRNA fragments directly lowers the purity and effective yield of full-length mRNA and may induce immunotoxicity. These fragments can act as reverse templates and participate in the generation of immunogenic dsRNA via non-specific transcription, further activating innate immune responses. Ensuring mRNA integrity is therefore essential to enhance drug potency and safety.
The industry adopts the following major strategies to mitigate mRNA fragment formation:
Transcriptase and promoter engineering: Genetic modification of RNA polymerases or fine-tuning of promoter sequences reduces the energy barrier for the transition from initiation complexes to elongation complexes, facilitating rapid entry into stable, high-efficiency transcription.
Supplementation of auxiliary factors: Addition of pyrophosphatase to transcription reactions consumes pyrophosphate byproducts, effectively lowering the incidence of abortive transcription and substantially boosting overall yields.
In-process control and anti-degradation measures: Incorporation of RNase inhibitors into IVT systems and rigorous control of metal ion contamination in production environments (metal ions accelerate phosphodiester bond hydrolysis) are necessary safeguards to prevent mRNA fragmentation at later stages. Furthermore, artificial intelligence algorithms can predict mRNA secondary structures, enabling sequence-level design to avoid motifs liable to trigger premature transcription termination as a forward-looking optimization approach.
For analytical detection, conventional methods exhibit limitations owing to the small size of mRNA fragments. Capillary gel electrophoresis (CGE) can assess full-length mRNA integrity yet lacks sufficient resolution for ultra-short abortive products. Currently, qualitative and quantitative analysis of these fragments predominantly relies on high-sensitivity liquid chromatography-mass spectrometry (LC-MS), which enables precise discrimination of fragments as short as several nucleotides and diverse complex impurities, supplying critical datasets for continuous process optimization.
mRNA Capping: Structural Integrity and Manufacturing Control
The 5′ capping of mRNA constitutes a pivotal determinant of its biological activity. Naturally occurring mRNA acquires a 7-methylguanosine (m⁷G) moiety at the 5′ terminus to form a specialized cap structure. This structure markedly improves mRNA stability against intracellular nuclease degradation and serves as the core recognition signal for ribosomes to initiate protein translation. Uncapped mRNA or mRNA bearing incorrectly oriented caps exhibits drastically diminished translation efficiency and undergoes rapid intracellular degradation, resulting in loss of therapeutic activity.
Two mainstream approaches are employed for mRNA capping in in vitro production: co-transcriptional capping, where cap analogues are supplemented directly into IVT reactions and incorporated onto the 5′ terminus of nascent mRNA during synthesis by RNA polymerases; and post-transcriptional capping, whereby mRNA undergoes terminal chemical modification via capping enzyme systems upon completion of IVT. Co-transcriptional capping is widely favored by the industry for its streamlined workflow and superior efficiency. Nevertheless, this approach faces challenges: cap analogues may undergo reverse incorporation, forming non-functional cap structures that hinder translation.
To address uncapped and mis-capped species, manufacturing processes adopt the following control strategies:
Employment of anti-reverse cap analogues (ARCA): Special chemical modification of the ribose moiety within cap analogues enforces incorporation in a single correct orientation, elevating the proportion of functionally capped mRNA.
Adoption of trinucleotide cap analogues: This currently represents the most efficient co-transcriptional capping strategy, enabling extremely high yields of native cap structures and delivering superior translational performance.
Optimization of reaction systems: Cap analogues compete with raw material GTP; tuning the molar ratio between the two maximizes capping efficiency while maintaining acceptable production yields.
In analytical testing, accurate differentiation between capped and uncapped mRNA lies at the heart of quality control. Due to the large molecular weight of mRNA, mainstream detection approaches generally require targeted pre-treatment:
Two-step enzymatic digestion coupled with high-performance liquid chromatography (HPLC): This represents the most cost-effective workflow. Specific nucleases are applied to degrade uncapped mRNA, followed by HPLC quantification of remaining capped products.
Liquid chromatography-mass spectrometry (LC-MS): mRNA is fragmented into short oligonucleotides prior to mass spectrometric detection, enabling high-sensitivity identification and supporting in-depth structural characterization.
PCR-based assays and nanopore sequencing: Quantitative ligase-mediated PCR and analogous techniques specifically identify uncapped fragments with distinct termini. As a cutting-edge technology, nanopore sequencing theoretically enables direct identification of methylated modifications at mRNA termini. With ongoing technical maturation, it is expected to realize intuitive characterization of capping integrity.
Removal of Process-Related Impurities and Purification Strategies
In addition to reaction byproducts originating from the target molecule itself, abundant process-related impurities remain in crude mRNA originating from IVT reaction mixtures, including residual DNA templates, unreacted nucleotide triphosphates (NTPs), T7 RNA polymerase, endotoxins, and various salts. These components are indispensable to support successful transcription and cannot be reduced during the reaction phase. Robust downstream purification is therefore mandatory to achieve efficient impurity removal and satisfy stringent safety and regulatory requirements.
Core Impurities and Associated Processing Challenges
DNA templates and RNA–DNA hybrids: DNA constitutes the highest-molecular-weight impurity in crude products. Excessive residual DNA may provoke inflammatory responses in vivo. DNase I digestion is conventionally used to fragment DNA templates. However, RNA–DNA hybrids generated during IVT are barely eliminated via standard DNase I treatment. These hybrids can activate innate immune sensors and pose potential risks of elevated product immunogenicity, demanding optimized chromatographic separation techniques.
Biogenic contaminants (endotoxins): Microbial expression systems such as Escherichia coli are commonly utilized to produce DNA templates and enzymes, which readily introduce bacterial lipopolysaccharides (endotoxins). Endotoxins exhibit potent immunogenicity and thermal stability. Strict quality control protocols must be enforced, including the use of endotoxin-free consumables and reagents, alongside compliant testing of final products to meet biopharmaceutical safety specifications.
Chromatography is the industry-preferred technology for scalable production of high-purity mRNA:
Oligo(dT) affinity chromatography: A well-established purification platform relying on base pairing between the poly(A) tail at the 3′ end of mRNA and immobilized oligo(dT) ligands. This method effectively removes unreacted nucleotides, salts and short RNA fragments to yield highly enriched target mRNA. Its primary limitation is the inability to deplete dsRNA impurities that also carry poly(A) tails, necessitating subsequent polishing steps.
Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC): A powerful polishing technique capable of substantial dsRNA depletion. However, widespread industrial adoption is hindered by reliance on toxic organic solvents and ion-pair reagents, alongside scalability constraints linked to cost and environmental compliance.
Anion exchange chromatography (AEX): An alternative candidate that separates mRNA and impurities based on charge differences. While demonstrating promising scalability, its capacity to resolve structurally analogous mRNA fragments and dsRNA awaits further exploration and validation.
It is evident that although current downstream workflows remove most impurities, there exists no universally established, platformized manufacturing process for mRNA pharmaceuticals comparable to that of monoclonal antibodies. Every available purification technique carries inherent technical trade-offs, such as the balance between throughput and impurity resolution. Future process development will focus on integrating orthogonal chromatographic technologies to reconcile cost, environmental requirements and product quality, thereby constructing more efficient and resilient mRNA manufacturing workflows.
Conclusion
The remarkable success of mRNA technology during epidemic response underscores its central role in modern medicine. Translating this potential into broad clinical applications hinges on overcoming the critical hurdle of high-quality mRNA production. Impurities generated during manufacturing, including dsRNA, truncated transcripts and uncapped mRNA species, not only suppress target protein translation efficiency but also elicit unintended immune responses in humans, directly impacting drug safety and therapeutic outcomes. Establishing a comprehensive framework for impurity identification, quantification and control is therefore fundamental to sustain the robust advancement of mRNA therapeutics.
