IVT mRNA and LNP-mRNA
VectorBuilder offers ready-to-use IVT mRNA and LNP encapsulated IVT mRNA that has been fully validated in vitro and in vivo, thus, it can be used as control for your mRNA experiment or to assess the efficiency of LNP-based mRNA delivery systems. For custom IVT mRNA and LNP preparation, check out VectorBuilder’s mRNA Gene Delivery Solutions. For scalable manufacturing of mRNA drugs, check out our CDMO services.
- Cap1 mRNA with validated UTRs and modified nucleotides, such as N1-Methylpseudouridine (m1Ψ)
- Sequence optimized HiExpressTM IVT mRNA for achieving robust expression
- Comprehensive and strict quality controls to ensure identity, purity, and functionality
Shipping and storage
Our mRNA products are stored in a 1 mM sodium citrate buffer (pH 6.4) and can be stored at -80°C for up to 12 months. LNP-mRNA products are stored in Tris buffer (pH 7.4) and can be stored at -80°C for up to 6 months. Both products are shipped on dry ice and should be avoided for freeze-thaw cycles.
Typical workflow of mRNA and LNP production
Figure 1. Typical workflow of mRNA synthesis and LNP packaging
Our mRNA production workflow starts with designing and synthesis of the template DNA sequence with the consideration of preferred codons, GC content, and thermodynamic stability of RNA secondary structures, followed by its cloning into an in vitro transcription vector. The plasmid DNA is then purified, validated, and linearized before being subsequently subjected to the in vitro transcription reaction which results in the generation of the desired transcript. Modified nucleotides can be incorporated into the in vitro transcription reaction to improve in vivo translation and decrease immunogenicity. Highly efficient capping (>95%) can be achieved either using co-transcriptional or enzymatic approaches. The mRNA is then purified by mRNA-capture beads as the default purification process or oligo dT chromatography upon request. Next, mRNA can be further encapsulated in LNP in a microfluidic mixer. The encapsulation efficiency and nanoparticle profiling are analyzed for LNP quality control.
Representative quality control (QC) results for IVT mRNA
Figure 2. Representative QC results of IVT mRNA.
(A) IVT mRNA integrity was assessed by running the sample on a denaturing agarose gel. A sharp band at expected size was observed. (B) IVT mRNA capping efficiency was analyzed by liquid chromatography-mass spectrometry (LC-MS). Area under the mass peaks in the deconvoluted mass spectrum was used to calculate the percentage of cap 1 structure.
Representative quality control (QC) results for LNP-mRNA
VectorBuilder’s QC tests for LNP-mRNA include measurements of various quality attributes to ensure the purity, efficacy, and stability. Default QC tests for the ready-to-use LNP-mRNA products include particle size, polydispersity index (PDI), zeta potential and encapsulation efficiency (EE%).
VectorBuilder has optimized our encapsulation technology to achieve a very low PDI (as low as PDI<0.1), additionally, VectorBuilder guarantees a zeta potential between -10 mV and +10 mV for all LNP-mRNA ensuring the homogeneity and efficacy of our products.
Figure 3. Representative QC results of LNP-mRNA.
(A) Particle size was determined by dynamic light scattering (DLS) which measures the intensity differences of fluctuated light due to motion of particles. The polydispersity index (PDI) reflects the heterogeneity of a sample on particle size. (B) Zeta potential reflects the stability of LNP. The Zeta potential of the sample is between -1.872 mV and +1.872 mV.
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What are the differences between mRNA caps and capping methods?
Cap 0 refers to N7-methylguanosine (m7G) that is added to the 5’ end of eukaryotic mRNAs via a 5’ to 5’ triphosphate linkage. This modification is added via a series of enzymatic reactions that occur co-transcriptionally and functions to regulate nuclear export, transcript stability, and promotes translation of the mRNA through recognition by eukaryotic translation initiation factor (eIF4E). Cap 1 refers to the addition of a methyl group to the 2’O on the first nucleotide (m7GpppNm) of the transcribed mRNA sequence in addition to the m7G cap. In mammalian cells, cap 1 structure is an important marker for mRNA to be recognized as self and not targeted by innate immunity. Adding cap 1 structure to synthesized mRNA has been demonstrated to enhance mRNA expression in vivo and reduced its immunogenicity.
Capping for in vitro transcribed RNA can occur either co-transcriptionally with cap analogs or post-transcriptionally via enzymatic reactions. The efficiency of our capping method has been well validated using LC-MS.
Why should I consider incorporating modified nucleotides in mRNA and which ones can be included?
Cells contain cytosolic and endosomal RNA receptors that activate the immune response upon recognition of foreign RNA. Modified nucleotides are commonly found in endogenous cellular RNA. Incorporating certain modified nucleotides in mRNA reduces its immunogenicity, alters secondary structure, and increases translation efficiency and half-life in a sequence-dependent manner. We provide the commonly used N1-Methylpseudouridine (m1Ψ). N1-Methylpseudouridine (m1Ψ) is a naturally occurring nucleotide that was first identified in tRNAs, however, its use in coding mRNAs has only recently been appreciated. This methylated derivative of uridine can replace the canonical nucleotide uridine in mRNA IVT and translation without altering traditional Watson-Crick base pairing. A major advantage of its use in mRNA therapeutics is its ability to alter recognition by RNA immune receptors thus mitigating unwanted immune effects and enhancing transcript stability and translation.