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Customizable, Accurate and Scalable Solutions
NJ Bio’s vast experience in bioconjugation and small molecules chemistry coupled with our robust technical and analytical platform allows us to carry out precise mRNA synthesis for diverse research applications. Our in vitro transcription (IVT) protocols and efficient purification methods are optimized to reduce dsRNA, residual proteins, and left-over reagents to deliver high integrity mRNA. We can synthesize custom NTPs for research and large-scale production of mRNA. Our state-of-the-art analytical laboratory meets the highest industry standards in characterization of mRNA for biopharmaceutical applications. Our scientists have in-depth experience in mRNA template design, which enables us to provide high-quality mRNA with excellent purity and biological activity to support the unique needs of our client’s drug discovery research and development program.
mRNA Service Options
Linearization of plasmid for transcription
In vitro transcription (IVT) from 100 microliters to multi-liters
Synthesis of natural & unnatural NTPs, capping agents, etc. on the gram scale
Process development, scale-up, GMP manufacturing
Co- or Post-transcriptional modifications
Incorporation of capping agent, natural and unnatural NTPs
All common UTP, ATP, GTP and CTP Analogs like m5U, m5C, m1A, 8-oxoG, m6Am available
5’ capping such as co-translational capping with Anti Reverse Cap Analog (ARCA)
Customization of Poly(A) tail length
Rapid, non-denaturing mRNA purification using IEC, TFF & affinity chromatography
Reverse-Phase HPLC, ELISA, construct-specific assays and method development
Potency assay, 5′-capping determination, analysis of poly(A) tail distribution
Quantification of residual protein/ DNA/ dsRNA, osmolality testing & measurement of endotoxin concentration
In the recent years there has been a widespread application of mRNA in the development of vaccines and cancer immunotherapy. To support the research and development of mRNA-based therapeutics, we offer a wide range of mRNA synthesis services that can be customized to fit the requirements of your research program. We can synthesize transcripts with a wide variety of modifications including 5′ terminal modifications using capping agents, internal modifications such as incorporation of nucleotide analogs like 5-methylcytidine (5mC) or pseudouridine (Ψ) and 3′ modification such as customization of poly(A) tail length.
G(5′)ppp(5’)G Cap Analog
Fig.1: Cap Analogs
NJ Bio’s mRNA synthesis capabilities include synthesizing research-grade mRNA using in vitro transcription (IVT) methods ranging from milligram to multi-gram scales with lengths ranging from a few hundred nucleotides to over 10 kilobases. Our mRNA transcripts are ideal for functional studies, library screening, preclinical studies and R&D of mRNA-based therapeutics and vaccines. Our research team provides consultative support throughout the design and optimization process and works collaboratively with our clients to synthesize the desired mRNA for their unique research applications.
Modified Nucleoside Triphosphates (NTPs)
The major advantage of incorporating modified nucleotides during IVT is improved translation and reduced immunogenicity. NJ Bio offers a wide selection of modified nucleoside triphosphates (NTPs) to suit your application needs. We can synthesize modified NTPs, including aminoallyl, biotin, 2′ fluoro, dye-labeled nucleotides, modified nucleotides such as alpha-thiol NTPs, and various base analogs.
Table 1. Commonly used modified nucleotides
Case Study 1: Large-scale synthesis of high purity Modified NTPs
NJ Bio entered the field of mRNA synthesis with the small-scale production of modified NTPs. Unlike most synthetic laboratories, NJ Bio’s strong background in bioconjugation empowers us with a robust technical and purification platform suited to synthesize these NTPs. Combining our expertise in bioconjugation and chemistry enables us to perform large-scale synthesis of modified NTPs to support our client’s mRNA programs. Presently, we can produce NTPs at a scale and purity level suitable for application in clinical trials or commercial production of mRNA. Our multi-disciplinary framework and collaborative approach helped us accelerate our client’s mRNA program and rapidly scale up NTPs from milligram to multi-gram scale within 6 months.
Case Study 2: Process optimization and scale up
Our NTP program expansion highlighted the similarity between the IVT reaction and enzymatic reactions routinely conducted in our laboratories. The connection prompted us to employ this technique for more efficient and faster scale-up. We developed a process that enabled us to scale up 100 microliters reaction to a 3 L reaction. Improvements in terms of temperature control, reagent sequence addition, and process optimization helped us achieve a more rapid and efficient scale up in a shorter period. Our synthetic ability enables us to produce high-quality reagents essential for scale up, in a short amount of time. We believe that we can further improve the efficiency of IVT – mRNA synthesis by proper reagent and solvent selection employing advanced purification techniques.
Case Study 3: Analytical Characterization
Analytical characterization of small and large molecules is an integral part of any mRNA or bioconjugation program, especially while producing clinical material. NJ Bio has a robust analytical platform to handle IVT analytics and product characterization. Our advanced instrumentation and technologies including HPLC, LC-MS, ELISA and CE were optimized for characterization of products and impurities generated during the process of mRNA synthesis. The development of this platform is underway and is expected to facilitate GMP-scale production by 2024 (H1). mRNA encapsulation and DNA production are other important additions that NJ Bio could offer after the planned expansion in 2024 (H1).
Nucleotide Synthesis using unmodified or Modified NTPs
There are more than 200 NTP types that can be used for diverse research applications. The standard or unmodified bases are the building blocks used for all IVT transcribed mRNA synthesis. Common modifications include base modified –, sugar modified –, and alpha-phosphate modified – NTPs.
Any modification to the nucleobase can improve the mRNA performance by reducing immunogenicity. Modifications to the ribose sugar at 2` hydroxyl position can impart RNA nuclease resistance. These modifications can also impact the yield of IVT transcription reactions. Modification to the phosphorothioate backbone by addition of thiophosphate at the alpha position can improve the rate of translation initiation. Addition of 1-Thio-ATP for polyadenylation prevents degradation of the poly(A) tail.
Nucleotide Synthesis (B=Modified/ Unmodified Base)
Fig.2 Process of Nucleotide Synthesis and Common NTP Modifications
IVT mRNA Production:
Our IVT mRNA synthesis procedure is simple yet versatile with multiple customizable options to suit unique research applications. NJ Bio can grow up to 2 L culture, i.e., up to 2-2.5 mg DNA for Low/ High copy plasmids. We can increase the culture volume to 20-30L and DNA content up to 100 mg using a bioreactor. We also use advanced technology and automated systems to ensure that the process of mRNA synthesis is error-free thus providing high-quality output.
Fig. 3 Process of in vitro synthesis of modified mRNA
NJ Bio offers the following mRNA services:
- Linearization of sequence-verified plasmid
- High-quality milligram to multigram synthesis of mRNA by IVT in 5L Glass Reactor and WAVE™ Reactors
- Plasmid cloning and purification
- Custom mRNA transcripts with modified or unmodified NTPs
- Identification, Characterization and Purification of Product using advanced MS and Reverse-Phase HPLC, Ӓkta Pilot systems
- Concentration measurement using SoloVPE technology
- Analysis of percent mRNA capped vs. uncapped
- Characterization of distribution of Poly(A) tail length
- Residual protein/ DNA/ dsRNA quantification, osmolality testing, potency assay and 5′-capping determination
- Endotoxin concentration measurement
- Faster purification of multiple grams of product in single run using the ÄKTA ready 450 Single-Use Chromatography System
- Full analytical capability for R&D to large scale manufacturing of mRNA
Frequently asked questions:
What is the purified synthetic yield of mRNA?
Custom mRNA is synthesized from milligram to multigram scale, in the range of 500 mg to 20 g. We generally aim to generate between 100X to 300X amplification of the sample based on the project requirements.
Which analytical procedures are used to process the final product?
We use state-of-the-art instrumentation to carry out reverse-phase HPLC, CGE and other construct specific assays. To know more about our bioanalytical services, visit https://njbio.com/bioanalytical-services-for-adcs-and-other-bioconjugates/
Which type of mRNA capping services are provided?
Standard mRNA transcripts are provided with mCAP or m7G(5′)ppp(5′)G capping. We additionally also offer Anti-Reverse Cap Analogs (ARCA) like 3´-O-Me-m7G(5′)ppp(5′)G.
What information do we need to submit to place a request for mRNA transcript synthesis?
We request you to provide information such as the desired mRNA sequence, 5′ and 3′ UTR sequence and details of any modified bases required.
How many A’s does the process of polyadenylation add to the tail?
Generally, mRNAs are produced with >100 nucleotide long poly(A) tail to enhance stability and translation efficiency.
How are the mRNA products shipped?
We typically ship the products as frozen mRNA solutions of >0.75 mg/ml concentrations in dry ice.