Poster
Analysis and Control of Poly(A) in mRNA Plasmids
Analysis and Control of Poly(A) in mRNA Plasmids
INTRODUCTION
Data
DNA
Primer
design
Primer
PCR ampl
-ification
Electroph
-oresis
Purification
analytics
extraction
synthesis
The global field of mRNA therapeutics is rapidly advancing, with a wide range of
downstream applications. As a critical raw material, the quality and especially the purity
of plasmid and linearized DNA directly influence the transcriptional efficiency and
translational output of in vitro transcribed (IVT) mRNA. Polyadenylic acid sequence, or
poly(A), plays an essential role in the stability, translational efficiency, and pharmacological
performance of mRNA. The length and integrity of poly(A) tails are considered critical
quality attributes (CQA) in the production of plasmid and linearized DNA. These
characteristics are receiving growing attention from researchers focused on analytical
methods and quality control strategies. Quality assessment of poly(A) is necessary
throughout cell banking, process development, and the production of plasmid and
linearized DNA.
Figure 4: Sanger sequencing flow chart
Capillary Gel Electrophoresis (CGE)
CGE separates DNA fragments based on differences in charge, size, and conformation
using a gel-filled capillary under high-voltage conditions. The gel matrix provides a
sieving effect, allowing high-resolution separation of nucleic acid fragments.
PLASMID PRODUCTION PROCESS
Cutting
Cutting
site2
site1
Poly(A) fragments
Linearized
Process
Plasmid
after enzyme digestion
03
04
01
02
DNA
Cell Banking
Development
production
production
Cutting site
CGE analysis
Figure 5：CGE method
Primary cell bank
(PCB)
Plasmid
Linearized DNA
production
Upstream process
development
Downstream process
development
production
ProBio has developed a CGE-based method to evaluate the integrity of the poly(A) tail.
This method offers high resolution, with the ability to resolve differences of 2 to 5 base
pairs. It also delivers excellent precision with a relative standard deviation below 4% and
high accuracy in the range of 90% to 110%.
Master cell bank
(MCB)
Quality standards
Quality standards
establishment
establishment
Testing and release
Working cell bank
(WCB)
Testing and release
Figure 1: Linearized DNA manufacturing process
Cell Banking
In cell banking, poly(A) analysis typically involves Sanger sequencing and capillary gel
electrophoresis (CGE). Due to the structure of mRNA plasmids, sequence identification must
evaluate both poly(A) and poly(T) signals. In most cases, poly(T) sequencing results are more
consistent and reliable. During CGE analysis, restriction enzymes are used to digest the
plasmid. Fragments containing the poly(A) tail are recovered and analyzed for distribution and
integrity. Based on the results, a clone is selected and used to establish the Primary Cell Bank
(PCB), Master Cell Bank (MCB), and Working Cell Bank (WCB).
Figure 6: CGE analysis
Liquid Chromatography–Mass Spectrometry (LC-MS)
LC-MS combines chromatographic separation with mass spectrometric identification
and is effective for high-resolution analysis of DNA fragments. Liquid chromatography
separates the sample for mass spectrometry’s identification. When applied to poly(A)
analysis, restriction enzymes are used to release DNA segments containing the
poly(A) tail.
Figure 2: Comparison of poly(A) and poly(T) sequencing signals
(The number of poly(T) is 117, and the number of poly(A) is 118.)
Process Development
These fragments are purified and analyzed based on their mass-to-charge ratios.
Enzyme site selection must account for the base sequence of the plasmid and the
detection range of the instrument.
Process development involves upstream fermentation and downstream purification. CGE is
used during both stages to monitor poly(A) purity. ProBio has extensive experience in
strain-specific fermentation optimization. The team also applies platform-based
chromatography systems and fine-tunes linearization conditions to preserve the stability of
the poly(A) tail.
Figure 7: LC-MS analysis
Comparison of the Three Methods
Pros&Cons
Method
The technology is well-established but cannot accurately
determine purity
Sanger
Figure 3: Poly(A) distribution from cell bank to process development
Can detect the distribution of different sizes but has a
5% discrepancy
CGE
Production and Release
After completing process development, pilot-scale production is initiated and evaluated
based on quality standards. Due to the structure and function of poly(A), both Sanger
sequencing and CGE are included in the release testing panel. Liquid chromatography–mass
spectrometry (LC-MS) can also be applied as a supplementary method. Increased purity of
the poly(A) tail in the linearized DNA is directly linked to improved mRNA purity and
performance following IVT process.
The resolution can reach up to 1 bp but the detectable
fragments are generally no larger than 150 bp
LC-MS
Table 1: Linearized DNA poly(A) purity affects mRNA purity
CONCLUSION
Item
Integrity of Poly(A) Purity of mRNA
Sanger sequencing and CGE are the primary analytical methods used to assess poly(A)
tail quality in mRNA plasmids. Sanger sequencing provides tail length and sequence
data, while CGE enables high-resolution profiling of tail integrity. LC-MS serves as an
advanced option for structural and distributional analysis where needed. Together, these
methods provide a comprehensive toolkit to monitor the critical quality attributes
(CQA) in mRNA plasmid development and manufacturing.
PROBIO PLASMID PLATFORM
ANALYTICAL METHODS FOR POLY(A) CHARACTERIZATION
Sanger Sequencing
ProBio’s CDMO platform supports all stages of drug development from preclinical
research to commercial production. Services include cell banking, process development,
and analytical method development, etc. The platform supports various applications
such as viral vector-based therapies, mRNA vaccines and drugs, Linear closed DNA
(LcDNA), and DNA-based therapeutics. The proprietary ProPlusTM linearized DNA
platform enhances plasmid purification and testing, reducing production timelines and
increasing output.
The Sanger sequencing, based on dideoxy chain termination, is a widely used technique. It
can be applied to both plasmid and linearized DNA. However, the repetitive nature of poly(A)
sequences often leads to signal dropout and high low-peak ratio. Polymerase chain reaction
(PCR) optimization and the template plasmid amount adjustment are commonly used to
improve the sequencing stability.
ProBioCDMO.com