Quality and Stability Testing of mRNA Drug Substances
- mRNA drug substances are capped by various methods after in vitro transcription using linearized plasmid DNA, etc. as a template.
- The poly(A) sequence of mRNA contributes to translation and intracellular stability, and the 5'-Cap structure shields the 5' end and confers resistance to exonucleases. Therefore, it is important to understand the structure and characteristics of mRNA in order to conduct its structural analysis and quality testing.
- A variety of analytical techniques are needed to evaluate impurities derived from the target substance, such as different chain lengths impurities that arise during manufacturing and double-stranded RNA (dsRNA), as well as impurities derived from the manufacturing process, such as E. coli genomic DNA, restriction enzymes, template DNA, and capping enzymes.
- We conduct quality testing and stability testing requested by our clients from the early research and development stage to the commercial stage in accordance with regulatory requirements, such as GMP and the Japan’s Reliability Criteria *1.
Operation system
- We conduct our studies based on ICH guidelines.
- We conform to the Japan’s Reliability Criteria*1, or GMP (Japan, the USA, and the Europe).
- We comply with Japanese and global laws, guidance, and guidelines, and we ensure data integrity*2.
Inspection and audit experience
- We have abundant experience in FDA inspections, PMDA inspections, QP audits, and audits by clients worldwide including Japan.
Features
- We develop analytical methods and conduct analytical method validation according to the most recent information, such as USP “Analytical Procedures for Quality of mRNA Vaccines and Therapeutics” (Draft Guidelines: 3rd Edition)*3.
- We conduct quality testing, stability testing, and release testing.
- We can also support our clients’ characterization of mRNA drug substances by LC/MS and subsequent establishment of specifications and test methods.
Quality tests and stability studies
- Long term testing, accelerated testing, stress testing, and photostability testing according to ICH Guidelines*4
- Stability storage facilities at our testing sites are monitored by centralized control systems (NASTER systems).
Samples handling
- We decontaminate the equipment and experimental benches in advance, to prevent contamination with ribonuclease, and conduct the tests in an RNase-free environment by wearing gloves and masks.
- We prevent contamination by zone-management, therefore, Reagents (controls) and samples (nucleic acid samples) are handled in the different laminar flow cabinets with regard to Sanger sequencing, real-time PCR and other tests.
- Powder containment facilities (EC2) enable our handling of highly potent compounds up to OEL Category 5.
- BSL-2 laboratories enable our handling of biohazard samples.
Test items
| Test items | Methods | Instruments | |
|---|---|---|---|
| Identity | Base sequence | Sanger sequence method | SeqStudio |
| LC/MS method | "Q Exactive plus Eclipse |
||
| Integrity | Full-length mRNA content | Agarose gel electrophoresis | E-Gel Power Snap |
| Microchip electrophoresis | Agilent 2100 Bioanalyzer | ||
| Capillary electrophoresis | PA 800 plus | ||
| Cap efficiency | LC/MS method, LC/UV methods | Q Exactive plus Eclipse, etc |
|
| Poly A chain length | LC/MS method, LC/UV methods | Q Exactive plus Eclipse, etc |
|
| Sanger sequence method | SeqStudio | ||
| Purity | dsRNA | Dot blot method | ChemiDoc XRS Plus |
| ELISA | "SpectraMax M5 SpectraMax iD5 |
||
| Residual DNA | Real-time PCR | QuantStudio 7 Flex | |
| Residual enzyme | ELISA | SpectraMax M5 SpectraMax iD5 |
|
| Quantitative determination | RNA content | Ultraviolet spectroscopy | NanoDrop Onec |
References
- *1: Article 43 of Regulation for Enforcement of the Act on Securing Quality, Efficacy and Safety of Products Including Pharmaceuticals and Medical Devices
- *2: FDA 21 CFR Part11
Notification by the Director General of the Pharmaceutical and Food Safety Bureau, MHLW Regarding Use of Electromagnetic Records and Electronic Signatures in Applications for Drug Approval or Licensing, etc. (PFSB Notification No. 0401022)
Annex11 to the EU Guide to GMP Computerized Systems
Notification " Guideline on Management of Computerized Systems for Marketing Authorization Holders and Manufacturers of Drugs and Quasi-drugs" (Pharmaceutical and Food Safety Bureau, Pharmaceutical and Food Safety Bureau, MHLW, PFSB Notification No. 1021-11)
A risk-based Approach to Compliant GxP Computerized Systems (GAMP 5), ISPE/GAMP COP (2008.2) - *3: Analytical Procedures for Quality of mRNA Vaccines and Therapeutics (Draft Guidelines: 3rd Edition)
https://www.uspnf.com/notices/analytical-procedures-mrna-vaccines-20240802 (accessed 2025.11.28) - *4: ICH Guideline, Stability Assay
https://www.pmda.go.jp/int-activities/int-harmony/ich/0038.html (accessed 2025.11.28)
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