Custom mRNA service from RUO to GMP

Accelerating your vaccine and drug development from discovery to production

Overview

mRNA (messenger RNA) is a promising substrate for novel therapeutics and has emerged as a leading candidate for vaccines, and therapies for cancers or genetic disorders. mRNA holds several unique properties that are advantageous in the development of new therapies, including highly localized expression patterns, rapid development time, and a high degree of safety as a non-genome editing substrate for therapies.

PackGene provides custom mRNA production with superior quality, industry-leading timelines, a wide range of deliverables, and stringent quality control. Our mRNA services can help to streamline the vaccine and drug development process and accelerate the translation of scientific discoveries into practical therapies by helping to smooth the transition from early research and development to GMP mRNA manufacturing.

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Superior Quality

  • Built-in CE test to guarantee mRNA quality
  • Guaranteed LNP particle size and uniformity
  • Comprehensive QC tests panel
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One-stop mRNA solution

  • On-shelf and custom mRNA
  • mRNA to LNP
  • RUO to cGMP
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Check out our Pre-designed, stocked reporter and genome-editing mRNAs and mRNA-LNP

How we make mRNA

How we make mRNA
*Included
Upon request

In vitro transcription (IVT) is a method used to synthesize mRNA in the laboratory using a DNA template and RNA polymerase enzymes. PackGene creates mRNA via IVT from plasmid templates containing a promoter sequence, your gene of interest, and a poly(A) tail. During IVT, the RNA polymerase enzymes recognizes the poly(A) tail sequence on the template and incorporates it into the mRNA. The use of plasmid templates for IVT with poly(A) tails can provide several benefits for mRNA production, including consistency, scalability, customization, and cost-effectiveness. These advantages make it a popular method mRNA production in research, preclinical, and clinical applications.

  • Simplified workflow:The incorporation of poly(A) tail during IVT directly from the plasmid template can simplify the workflow since there is no need for an additional step to add the poly(A) tail post-transcription. This in turn shortens total production time.
  • Higher efficiency: Incorporating poly(A) tail during IVT directly from the plasmid template can be more efficient than adding it after transcription, as there is a greater likelihood of polyadenylation of the mRNA due to the presence of a poly(A) tail sequence on the template.
  • Consistency: Using plasmid templates with a pre-existing poly(A) tail sequence can ensure consistency in the length and composition of the poly(A) tail across batches of mRNA produced.
  • Scalability: The use of plasmid templates can also enable the scalable production of mRNA. Once the plasmid template is established, the production of mRNA can be easily scaled up to produce large quantities of mRNA for research, preclinical, or clinical use.
  • Customization: Plasmid templates can be engineered to contain specific regulatory elements, such as promoters and enhancers, which can be used to control the expression and regulation of the mRNA. Additionally, the use of poly(A) tails can enhance the stability of the mRNA, which can improve its translation efficiency and expression levels.
  • Cost-effectiveness: Using plasmid templates for IVT can be a cost-effective method for mRNA production. The cost of plasmid DNA synthesis is relatively low, and the use of poly(A) tails can eliminate the need for expensive poly(A) tailing enzymes.
What We Offer
Catalog mRNA and LNP Product Gene Quantity Timeline (Business days)
mRNA On-shelf mRNA Cap1 EGFP / mCherry / Firefly Luciferase / SpCas9 100μg-1mg 1-3
On-shelf mRNA Cap1 N1meΨU 1-3
Custom mRNA Cap1 Custom gene 10-15
Custom mRNA Cap1 N1meΨU 10-15
mRNA-LNP On-shelf mRNA Cap1 in LNP EGFP / mCherry / Firefly Luciferase / SpCas9 100μg-2mg 5-12
On-shelf mRNA Cap1 N1meΨU in LNP 5-12
Custom mRNA Cap1 in LNP Custom gene 15-25
Custom mRNA Cap1 N1meΨU in LNP 15-25

*Additional time (~2-3 weeks ) for custom gene synthesis

mRNA

mRNA grade and QC standard

QC Category QC Item Method Specification Research Grade Add-on
Identification Appearance Visual Inspection Clear and free of foreign particles
mRNA Concentration UV Absorbance by Nanodrop ≥1mg/ml
RNA Integrity/size Capillary Electrophoresis Target ±30%, Expected band size detected
Buffer Specification Client Spec RnaseFree H2O(default), PBS, 1mM Sodium Citrate, pH 6.4
Purity A 260/280 Ratio UV Absorbance by Nanodrop 1.70-2.30
Size based purity Capillary Electrophoresis >80%
Impurity Total Protein residue Nano Orange ≤1%
Plamsid DNA residue qPCR ≤0.1%
dsRNA Slot-blot ≤1%
Safety Endotoxin Semiquantitative LAL <10EU/mg
Endotoxin Quantitative <10EU/mg
Bioburden Direct Inoculation No growth after 48 hrs
Raw material-Linearization Linearization percentage AGE >95% HPLC
Host cell DNA Quantitative PCR ≤ 5%
Total protein Nano Orange ≤1%
RNA AGE Non-detectable by gel electrophoresis at 200ng
Raw material -circular Plasmid Gene of Interest Sanger sequencing 100% math reference sequence (not including poly A)
Poly A Sanger sequencing ≤110A ±5nt, ≤111-125A ±8nt
Poly A Length Enzyme digestion and CE Target ±5%
 

(Scroll down to see more)

In vitro transcribed (IVT) mRNA is a crucial component in the development of mRNA-based therapeutics and vaccines. To ensure the quality of IVT mRNA, several tests can be performed. Here are some important tests to measure the quality of IVT mRNA:

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Gel electrophoresis

This is a technique used to separate and visualize RNA fragments based on their size. It can be used to confirm the integrity of the mRNA, as well as the absence of any degradation or impurities.

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Spectrophotometry

This is a technique used to measure the concentration of RNA in a solution. It can be used to ensure that the mRNA concentration is within the desired range and that there are no contaminants that can interfere with downstream applications.

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Capillary electrophoresis

This is a technique used to measure the purity of mRNA by separating RNA fragments based on their size and charge. It can be used to determine the percentage of full-length mRNA, as well as the presence of truncated or degraded fragments.

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Endotoxin testing

Endotoxins are lipopolysaccharides that can cause adverse immune reactions in humans. To ensure the safety of IVT mRNA, endotoxin levels should be measured and kept below a certain threshold.

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Sterility testing

To ensure that the IVT mRNA is free of any microbial contamination, sterility testing can be performed using standard microbiological techniques.

Additional tests can be added to further verify the quality of the mRNA

  • Total protein by Nano Orange: This test is used to measure the total protein content in the IVT mRNA sample. The Nano Orange dye binds to proteins in the sample, and the fluorescence signal is measured to determine the protein concentration. This test can help ensure that the mRNA sample is free of protein contaminants, which can interfere with downstream applications.
  • Plasmid DNA residue by qPCR: This test is used to measure the residual plasmid DNA in the IVT mRNA sample. Plasmid DNA is often used as a template for mRNA synthesis, and residual plasmid DNA can remain in the final product, which can trigger an immune response in the host. qPCR is a sensitive technique that can detect even low levels of plasmid DNA in the mRNA sample.
  • dsRNA by slot-blot: This test is used to measure the presence of double-stranded RNA (dsRNA) in the IVT mRNA sample. dsRNA can be formed during the mRNA synthesis process and can trigger an immune response in the host. Slot-blot is a technique used to transfer RNA samples onto a nitrocellulose membrane, which is then hybridized with a dsRNA-specific probe. The resulting signal can be quantified to determine the amount of dsRNA in the sample.

 

Overall, these tests can help ensure the quality, purity, and safety of IVT mRNA, which are essential for its successful use in mRNA-based therapeutics and vaccines.

Transfection efficiency of mRNA to cell lines

72 hrs after transfection

Firefly Luciferase mRNA after transfection

EGFP mRNA(Cap1)
2μg 24hrs after transfection

EGFP mRNA after transfection

mCherry mRNA(Cap1)
2μg 24hrs after transfection

mCherry mRNA after transfection
mRNA-Lipid Nanoparticle (LNP)

Lipid nanoparticles (LNPs) are nanoscale particles composed of lipids and are used as delivery vehicles for drugs, including mRNA. LNP based methods are the most popular methods for mRNA delivery and offer several benefits, including increased stability, increased delivery efficiency, reduced immunogenicity, customizability, and a history of clinical success. These advantages make LNPs a promising tool for the development and application of safe and effective mRNA-based treatments and vaccines.

  • Increased stability: LNPs can protect mRNA from degradation by enzymes and other factors in the body, allowing for increased stability and improved delivery of the mRNA to the target cells.
  • Enhanced delivery: LNPs can facilitate more efficient uptake of mRNA by cells, leading to higher levels of protein expression. LNPs can also drive targeted delivery of mRNA to specific cells, such as immune or tumor cells, by enabling uptake through cell-type specific endocytosis mechanisms.
  • Reduced immunogenicity: LNPs can help reduce the immunogenicity of mRNA by shielding the mRNA from the immune system and preventing the activation of innate immune responses. This can improve the safety and efficacy of mRNA-based therapies and vaccines.
  • Customization: The composition of LNPs can be modified to improve their properties, such as stability, delivery efficiency, and immunogenicity. This allows for customization of LNPs toward specific applications, such as vaccines or therapies targeting specific diseases.
  • Clinical success: LNPs have been used successfully in the development of mRNA vaccines for COVID-19, showing high efficacy and safety in clinical trials. This success has increased interest in the use of LNPs for other mRNA-based therapies and vaccines.
mRNA
mRNA-LNP QC Control
QC category QC item Method Specification Research Grade
mRNA-LNP
(On-shelf and Custom)
RNA Integrity/size Capillary Electrophoresis >80%
Encapsulate Efficiency Elisa Analyzer Report
Particle size Dynamic Light Scattering <120nm
Particle Uniformity Dynamic Light Scattering <0.2
Concentration Capillary Electrophoresis Encapsulated mRNA <0.2mg/ml

PackGene’s quality control (QC) testing of mRNA lipid nanoparticles (LNPs) includes measurement of various parameters to ensure their quality, safety, and efficacy. Some of the essential QC tests for mRNA LNP are encapsulation efficiency, particle size, and uniformity.

  • Encapsulation Efficiency: Encapsulation efficiency (EE) is a measure of how much mRNA is encapsulated within the lipid nanoparticles (LNP). High EE is crucial for ensuring that an adequate amount of mRNA is delivered to the target cells, as a lower EE can result in a reduced therapeutic effect. EE can be measured by separating the free mRNA from the LNPs using a centrifugation or ultrafiltration method, followed by quantification of the encapsulated mRNA. Measurement of encapsulation efficiency is carried out over several steps; the steps are as follows:
    1. Obtain the total amount of mRNA used in the formulation. This can be determined by measuring the concentration of the mRNA solution and multiplying it by the total volume used.
    2. Separate the encapsulated mRNA from the free mRNA. This is commonly done by centrifugation or ultrafiltration.
    3. Measure the amount of encapsulated mRNA. This can be determined by quantifying the concentration of mRNA in the pellet after demulsification.
    4. Calculate the encapsulation efficiency using the following formula:
      • Encapsulation Efficiency (EE) = (Amount of encapsulated mRNA / Total amount of mRNA) x 100%

        For example, if you used 1 mg of mRNA in the formulation and obtained 0.8 mg of encapsulated mRNA after centrifugation, the encapsulation efficiency would be:

        EE = (0.8 mg / 1 mg) x 100% = 80%

        This means that 80% of the mRNA was encapsulated within the LNP, and the remaining 20% was free in solution.

  • Particle Size: Particle size is a critical parameter for the efficacy and safety of LNPs. Particle size affects the biodistribution and cellular uptake of LNPs, and larger particles may be more prone to aggregation, leading to decreased stability and increased toxicity. Particle size can be measured using dynamic light scattering (DLS), which determines the hydrodynamic diameter of the particles.
  • Size Distribution and Uniformity: In addition to particle size, the size distribution and uniformity of LNPs are important for their stability and efficacy. A narrow size distribution and uniformity can improve the biodistribution and cellular uptake of LNPs, whereas a broad size distribution can lead to variable uptake and potential toxicity. The size distribution and uniformity can be measured using DLS or other methods, such as nanoparticle tracking analysis (NTA).

 

These parameters are critical for determining the efficacy of the LNPs in delivering mRNA to the target cells and tissues, and for assessing the potential for toxicity and adverse effects.

Case Study

IVT mRNA performance

EGFP mRNA (Cap1)

EGFP mRNA

Firefly Luciferase mRNA (Cap1)

Firefly Luciferase mRNA

mCherry mRNA (Cap1)

mCherry mRNA

SpCas9 mRNA (Cap1)

SpCas9 mRNA

mRNA produced by PackGene shows the size of the mRNA falls within a 30% range of the targeted size and >80% purity. +/-30% targeted size ensures that the mRNA is of the appropriate size for the intended application and that there is consistency in the size of the mRNA between different batches of samples. A purity of >80% indicates that the sample has a high level of mRNA content, and relatively low levels of contaminants, such as genomic DNA, RNA degradation products, and residual reagents from the mRNA synthesis process which can affect the efficacy and safety of the mRNA in downstream applications.

The datas are measured by Agilent 5200 CE System, a highly sensitive and precise instrument for the analysis of nucleic acids, including mRNA.

EGFP mRNA (Cap1) -LNP

mass distribution
intensity distribution
Z Ave. Dia (nm) 76.8
PDI PDI
Encapsulation Efficiency 93.91%
The Z average diameter is determined by measuring the Brownian motion of the nanoparticles using dynamic light scattering (DLS) technology. During DLS analysis, the nanoparticles in solution are exposed to a laser beam, and the scattered light is measured at different angles. The rate of Brownian motion of the nanoparticles is then calculated based on the measured fluctuations in the scattered light intensity, which can be used to determine the size distribution of the particles in solution.

The Z average diameter is calculated as the intensity-weighted mean of the size distribution, taking into account the number of particles and their intensity (scattering power). This means that larger particles will contribute more to the overall Z average diameter than smaller particles. The Z average diameter is often reported in nanometers (nm) and is an important parameter for characterizing the size of nanoparticles, as it reflects the overall size of the population of particles in solution. mRNA-LNP produced by PackGene has a guarantee of Z average diameter <120nm and encapsulation efficiency of >90%

PDI stands for Polydispersity Index, which is a measure of the uniformity or heterogeneity of particle sizes within a sample of nanoparticles, including lipid nanoparticles (LNPs) used in mRNA delivery. It is a measure of the breadth of the size distribution curve of the nanoparticles. A low PDI value indicates that the nanoparticles in the sample have a relatively narrow size distribution, while a high PDI value indicates that the sample contains nanoparticles with a wide range of sizes.

In the context of mRNA delivery, a low PDI value is desirable because it indicates that the LNPs in the sample are relatively uniform in size, which can improve their stability, efficacy, and safety in vivo. A high PDI value may be indicative of poor LNP formulation or manufacturing, which can lead to variable particle sizes and affect the consistency and reproducibility of the mRNA delivery system. mRNA-LNP produced by PackGene has a guarantee of PDI <0.2

Injection of mRNA-LNP to animals

Firefly luciferase mRNA9(Cap1)-LNP
Live image 6hrs after mouse intravenous injection

LNP+Firefly Luciferase mRNA

EGFP mRNA9(Cap1)-LNP
Confocal image of mouse intestine frozen section 24hrs after enema

LNP+EGFP mRNA
Application

Messenger RNA (mRNA) is a type of RNA that carries genetic information from DNA to ribosomes, where it is translated into protein. In recent years, mRNA technology has gained significant attention due to its potential applications in various fields. Some of the key applications of mRNA include:

Vaccine
Development
Therapeutics
Cellular
Reprogramming
Resources
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