Imagine a future where rare diseases and devastating cancers can be swiftly treated with therapies produced directly in the clinic and tailored to a patient’s unique genetic profile. Common conditions could be managed with medicines that target their underlying causes with molecular precision. Infectious diseases could be controlled as soon as the pathogen genome was sequenced.
That future is starting now thanks to RNA therapeutics, a rapidly evolving class of innovative drugs offering personalized solutions across a range of conditions, from cardiovascular disease to cancer.
James M. Coffin, PhD
Currently, more than twenty RNA-based therapies are FDA-approved, with hundreds more in clinical trials.1 This number is rising exponentially as artificial intelligence accelerates the design and development of RNA therapeutics. However, RNA manufacturing practices have not kept pace.
Traditional RNA production relies on batch in vitro transcription (IVT), which is relatively slow, expensive, and inflexible with regards to scale of production, typically designed to serve pandemic-level needs. As the demand for RNA drugs grows, it is vital to modernize the biomanufacturing process to expedite their delivery to patients.
NTx, a New Mexico-based biotech company specializing in biomanufacturing solutions for RNA, protein, and critical raw materials, is focused on delivering a new approach to manufacturing that moves at the same speed as RNA innovation. To that end, we describe here the use of NTxscribe®, a benchtop, continuous flow IVT system that integrates synthesis and purification in a closed, modular system to increase RNA manufacturing efficiency.
The limitations of traditional batch RNA manufacturing can significantly delay getting therapies from the bench to the bedside. Logistically, batch production is slow, complex, and fragmented, requiring coordination across multiple vendors for materials, specialized purification protocols, and specialized facilities with experienced bioprocessing teams. This lack of a unified support system increases the risk of variability in RNA quality and leaves manufacturers vulnerable to supply chain disruptions, as seen during the global scramble for vaccine production resources in 2020-2021.
Moreover, batch RNA production is slow, taking six to eight months to develop and optimize. Each component process, such as IVT, purification, and formulation, demands rigorous adjustment, as variations can compromise quality and yield. These delays can hinder time-sensitive applications, such as emergent infectious disease responses or individualized cancer therapies and make it costly to advance new therapeutics—especially ones designed for a single patient.
Batch IVT involves mixing DNA template constructs, nucleotides, enzymes, and other reagents in one large volume reaction vessel, which is then incubated and agitated to facilitate transcription. The excess reagents and agitation stresses produce RNA prone to integrity loss and contamination, especially for longer sequences. Following synthesis, the resulting RNA must be extracted and purified prior to final formulation, often requiring lithium chloride precipitation and chromatography.
These additional steps may introduce variability, prolong process development, and compromise integrity. Scaling is complicated and expensive, often requiring an entire process redesign for each incremental scale-up. These inefficiencies create bottlenecks that slow production, increase costs, and can make the development of novel RNA therapeutics inaccessible or unsustainable.
To address the above limitations, NTxscribe uses an integrated, continuous flow design that offers cell-free RNA synthesis with minimal handling requirements. Its hollow fiber bioreactor (HFBR) (Figure 1) maintains steady-state reaction conditions and minimizes shear stress, facilitating linear high-yield, scalable synthesis with low contaminating dsRNA content. Following IVT, RNA is transferred directly into an inline purification cartridge that eliminates the need for lithium chloride precipitation or organic solvents.
![As the demand for RNA drugs grows, it is vital to modernize the biomanufacturing process to expedite their delivery to patients. [NTx]](https://www.genengnews.com/wp-content/uploads/2025/06/NTx-Lab-200x300.jpg "As the demand for RNA drugs grows, it is vital to modernize the biomanufacturing process to expedite their delivery to patients. [NTx]")
As the demand for RNA drugs grows, it is vital to modernize the biomanufacturing process to expedite their delivery to patients. [NTx]
The system can produce 25 to 60+ mg of high-integrity purified messenger RNA (mRNA) or self-amplifying RNA (saRNA) in under three hours. This contrasts sharply with the six to eight months required to develop and optimize batch RNA production. The system also enables linear scaling from small research quantities to commercial production using the same process, making it considerably more agile than batch methods, which are designed for high-volume output of millions of identical doses and unsuited to small-volume, patient-specific needs.
Moreover, single use, closed cassettes are designed for accessibility across both preclinical and clinical settings. For therapeutic applications, the company offers efficient incorporation of modified nucleotides such as N1-methylpseudouridine, which enhance RNA stability and reduce immunogenicity.
By applying continuous flow principles to RNA synthesis and purification, the biggest pain points in IVT, including speed, scalability, and cost are addressed. The platform operates as a closed system with single-use IVT and Purification cartridges that enable immediate production changes, eliminating the labor-intensive cleaning and resetting routines associated with batch methods (Figure 1):
Figure 1. The NTxscribe bioreactor and workflow
Together, these modules provide an uninterrupted path from DNA template to mRNA and saRNA, delivering a product suitable for research-use-only (RUO) and, in the future, good manufacturing practice (GMP) settings and compatible with standard delivery modalities such as cell-based therapeutics and nanoparticle encapsulation.
Standard biomanufacturing processes must be modernized to meet the growing demand for RNA therapeutics. NTxscribe was developed to address this need and, in proof-of-concept studies, has emerged as an efficient and viable option for researchers and companies requiring RNA production flexibility.
Figure 2. Experimental Results: Synthesis of complex saRNA on the NTxscribe IVT module. a) Integrity Electropherogram comparing conventional manufacturing (reference; red) with NTxscribe production with 7-methylguanosine (+m7G) and without (-m7G). b) BHK-21 cell expression.
In a COVID-19 vaccine candidate study, the system synthesized 85 milligrams of high-quality co-transcriptionally capped saRNA in just 40 minutes (Figure 2). The integrity and functional expression of this 11,500 nucleotide saRNA sequence was superior (3.5x higher in vitro expression) to that of purified internal pharmaceutical reference standards, even without additional downstream purification. This performance offers an alternative to conventional methods that require extensive purification and longer timelines.
Independent third-party testing by investigators at Houston Methodist Hospital further confirmed the platform’s performance (Figure 3). Electropherogram analysis revealed clean mRNA profiles with minimal impurities, while ELISA assays showed dsRNA levels at 0.2% to 0.35%, well within accepted therapeutic thresholds. Low levels of dsRNA and other contaminants minimize the risk of unwanted immune responses.
Figure 3. Independent validation of mRNA integrity produced by NTxscribe, performed by Dr. John Cooke’s laboratory (Houston Methodist Hospital). (a) Electropherogram of Cas9 mRNA showing a clean profile with minimal impurities after LiCI precipitation (b) Electropherogram of firefly luciferase (fLuc) mRNA, again showing a sharp peak with limited impurities, indicating intact sequences. (c) TapeStation RNA analysis confirming expected transcript sizes for both Cas9 and fLuc mRNA. All transcripts were co-transcriptionally capped, polyadenylated, and incorporated with N1MePsU. d) dsRNA quantification via ELISA for Cas9 and fLuc.
The system has also been adopted by a leading U.S. cancer center for producing personalized oncology vaccines. Researchers at this institute are leveraging NTxscribe to develop saRNA constructs that encode tumor antigens, enabling on-site therapeutic development.
Continuous-flow IVT provides a technical path toward decentralized and scalable RNA production. The ability to rapidly generate high-quality RNA without relying on centralized manufacturing infrastructure enables more agile development timelines, particularly for personalized applications. NTxscribe is designed to enhance reproducibility at both small and large scales, particularly for long and complex sequences.
The platform’s adaptability lends itself to both genetic disease research and rare disease applications, with its ability to deliver milligram- to gram-scale output enabling it to serve both exploratory and clinical-stage projects. NTxscribe also supports the generation of saRNA-based cancer vaccines, which can be customized for individual patients.
Continuous flow technology is a paradigm shift in RNA production, enabling researchers and companies to adapt their production today and lead RNA-based medicine tomorrow. While batch IVT methods are increasingly misaligned with the fast pace and agility of RNA therapeutic innovation, an integrated continuous flow approach enables flexible, high-quality RNA production in minutes rather than months and expands access to affordable, next generation bioprocessing for personalized therapies across the spectrum of health and disease.
Reference
1Avalere. (2024) Overview and Outlook for RNA-Based Therapies [White paper]. https://avalere.com/wp-content/uplo...ly-RNA-Based-Therapies-White-Paper-vFINAL.pdf
James M. Coffin, PhD, is president and CEO of NTx.
The post Modernizing RNA Therapeutic Manufacturing with Continuous Flow <i>In Vitro</i> Transcription appeared first on GEN - Genetic Engineering and Biotechnology News.
That future is starting now thanks to RNA therapeutics, a rapidly evolving class of innovative drugs offering personalized solutions across a range of conditions, from cardiovascular disease to cancer.
James M. Coffin, PhD
Currently, more than twenty RNA-based therapies are FDA-approved, with hundreds more in clinical trials.1 This number is rising exponentially as artificial intelligence accelerates the design and development of RNA therapeutics. However, RNA manufacturing practices have not kept pace.
Traditional RNA production relies on batch in vitro transcription (IVT), which is relatively slow, expensive, and inflexible with regards to scale of production, typically designed to serve pandemic-level needs. As the demand for RNA drugs grows, it is vital to modernize the biomanufacturing process to expedite their delivery to patients.
NTx, a New Mexico-based biotech company specializing in biomanufacturing solutions for RNA, protein, and critical raw materials, is focused on delivering a new approach to manufacturing that moves at the same speed as RNA innovation. To that end, we describe here the use of NTxscribe®, a benchtop, continuous flow IVT system that integrates synthesis and purification in a closed, modular system to increase RNA manufacturing efficiency.
The batch bottleneck
The limitations of traditional batch RNA manufacturing can significantly delay getting therapies from the bench to the bedside. Logistically, batch production is slow, complex, and fragmented, requiring coordination across multiple vendors for materials, specialized purification protocols, and specialized facilities with experienced bioprocessing teams. This lack of a unified support system increases the risk of variability in RNA quality and leaves manufacturers vulnerable to supply chain disruptions, as seen during the global scramble for vaccine production resources in 2020-2021.
Moreover, batch RNA production is slow, taking six to eight months to develop and optimize. Each component process, such as IVT, purification, and formulation, demands rigorous adjustment, as variations can compromise quality and yield. These delays can hinder time-sensitive applications, such as emergent infectious disease responses or individualized cancer therapies and make it costly to advance new therapeutics—especially ones designed for a single patient.
Batch IVT involves mixing DNA template constructs, nucleotides, enzymes, and other reagents in one large volume reaction vessel, which is then incubated and agitated to facilitate transcription. The excess reagents and agitation stresses produce RNA prone to integrity loss and contamination, especially for longer sequences. Following synthesis, the resulting RNA must be extracted and purified prior to final formulation, often requiring lithium chloride precipitation and chromatography.
These additional steps may introduce variability, prolong process development, and compromise integrity. Scaling is complicated and expensive, often requiring an entire process redesign for each incremental scale-up. These inefficiencies create bottlenecks that slow production, increase costs, and can make the development of novel RNA therapeutics inaccessible or unsustainable.
Continuous-flow RNA production using a hollow-fiber bioreactor
To address the above limitations, NTxscribe uses an integrated, continuous flow design that offers cell-free RNA synthesis with minimal handling requirements. Its hollow fiber bioreactor (HFBR) (Figure 1) maintains steady-state reaction conditions and minimizes shear stress, facilitating linear high-yield, scalable synthesis with low contaminating dsRNA content. Following IVT, RNA is transferred directly into an inline purification cartridge that eliminates the need for lithium chloride precipitation or organic solvents.
As the demand for RNA drugs grows, it is vital to modernize the biomanufacturing process to expedite their delivery to patients. [NTx]
The system can produce 25 to 60+ mg of high-integrity purified messenger RNA (mRNA) or self-amplifying RNA (saRNA) in under three hours. This contrasts sharply with the six to eight months required to develop and optimize batch RNA production. The system also enables linear scaling from small research quantities to commercial production using the same process, making it considerably more agile than batch methods, which are designed for high-volume output of millions of identical doses and unsuited to small-volume, patient-specific needs.
Moreover, single use, closed cassettes are designed for accessibility across both preclinical and clinical settings. For therapeutic applications, the company offers efficient incorporation of modified nucleotides such as N1-methylpseudouridine, which enhance RNA stability and reduce immunogenicity.
By applying continuous flow principles to RNA synthesis and purification, the biggest pain points in IVT, including speed, scalability, and cost are addressed. The platform operates as a closed system with single-use IVT and Purification cartridges that enable immediate production changes, eliminating the labor-intensive cleaning and resetting routines associated with batch methods (Figure 1):
- IVT cartridge: Contains an HFBR that produces high-integrity RNA, with the ability to adapt production from milligram to gram scale synthesis runs. Unlike batch systems that start and stop, the HFBR maintains a constant flow, supporting continuous enzyme activity and efficient nucleotide incorporation. For mRNA, co-transcriptional capping (99% efficient) and template-encoded poly(A) tails are utilized to ensure accurate production of capped and tailed RNA.
- RNA transfer: The rapid seamless flow of RNA from the IVT module cartridge into the Purification cartridge minimizes the risk of contamination and degradation. This continuous flow manufacturing also removes the need for infrastructure-heavy processes and multiple specialist operators.
- Purification cartridge: Uses continuous flow purification to remove contaminants such as double-stranded RNA (dsRNA) and protein residues to ensure high purity and quality without the use of precipitation methods and organic solvents.
Figure 1. The NTxscribe bioreactor and workflow
Together, these modules provide an uninterrupted path from DNA template to mRNA and saRNA, delivering a product suitable for research-use-only (RUO) and, in the future, good manufacturing practice (GMP) settings and compatible with standard delivery modalities such as cell-based therapeutics and nanoparticle encapsulation.
Performance and validation
Standard biomanufacturing processes must be modernized to meet the growing demand for RNA therapeutics. NTxscribe was developed to address this need and, in proof-of-concept studies, has emerged as an efficient and viable option for researchers and companies requiring RNA production flexibility.
Figure 2. Experimental Results: Synthesis of complex saRNA on the NTxscribe IVT module. a) Integrity Electropherogram comparing conventional manufacturing (reference; red) with NTxscribe production with 7-methylguanosine (+m7G) and without (-m7G). b) BHK-21 cell expression.
In a COVID-19 vaccine candidate study, the system synthesized 85 milligrams of high-quality co-transcriptionally capped saRNA in just 40 minutes (Figure 2). The integrity and functional expression of this 11,500 nucleotide saRNA sequence was superior (3.5x higher in vitro expression) to that of purified internal pharmaceutical reference standards, even without additional downstream purification. This performance offers an alternative to conventional methods that require extensive purification and longer timelines.
Independent third-party testing by investigators at Houston Methodist Hospital further confirmed the platform’s performance (Figure 3). Electropherogram analysis revealed clean mRNA profiles with minimal impurities, while ELISA assays showed dsRNA levels at 0.2% to 0.35%, well within accepted therapeutic thresholds. Low levels of dsRNA and other contaminants minimize the risk of unwanted immune responses.
Figure 3. Independent validation of mRNA integrity produced by NTxscribe, performed by Dr. John Cooke’s laboratory (Houston Methodist Hospital). (a) Electropherogram of Cas9 mRNA showing a clean profile with minimal impurities after LiCI precipitation (b) Electropherogram of firefly luciferase (fLuc) mRNA, again showing a sharp peak with limited impurities, indicating intact sequences. (c) TapeStation RNA analysis confirming expected transcript sizes for both Cas9 and fLuc mRNA. All transcripts were co-transcriptionally capped, polyadenylated, and incorporated with N1MePsU. d) dsRNA quantification via ELISA for Cas9 and fLuc.
The system has also been adopted by a leading U.S. cancer center for producing personalized oncology vaccines. Researchers at this institute are leveraging NTxscribe to develop saRNA constructs that encode tumor antigens, enabling on-site therapeutic development.
Implications for research and therapeutic development
Continuous-flow IVT provides a technical path toward decentralized and scalable RNA production. The ability to rapidly generate high-quality RNA without relying on centralized manufacturing infrastructure enables more agile development timelines, particularly for personalized applications. NTxscribe is designed to enhance reproducibility at both small and large scales, particularly for long and complex sequences.
The platform’s adaptability lends itself to both genetic disease research and rare disease applications, with its ability to deliver milligram- to gram-scale output enabling it to serve both exploratory and clinical-stage projects. NTxscribe also supports the generation of saRNA-based cancer vaccines, which can be customized for individual patients.
Continuous flow technology is a paradigm shift in RNA production, enabling researchers and companies to adapt their production today and lead RNA-based medicine tomorrow. While batch IVT methods are increasingly misaligned with the fast pace and agility of RNA therapeutic innovation, an integrated continuous flow approach enables flexible, high-quality RNA production in minutes rather than months and expands access to affordable, next generation bioprocessing for personalized therapies across the spectrum of health and disease.
Reference
1Avalere. (2024) Overview and Outlook for RNA-Based Therapies [White paper]. https://avalere.com/wp-content/uplo...ly-RNA-Based-Therapies-White-Paper-vFINAL.pdf
James M. Coffin, PhD, is president and CEO of NTx.
The post Modernizing RNA Therapeutic Manufacturing with Continuous Flow <i>In Vitro</i> Transcription appeared first on GEN - Genetic Engineering and Biotechnology News.