White Papers
Challenges and Solutions in the Poly(A) Control for Plasmid & mRNA
Recent advances in mRNA therapeutics
and vaccines have enabled novel approaches to
combat cancer, genetic disorders, infectious
diseases,
and
more.
The
therapeutic
effectiveness of mRNA relies heavily on its
ability to be stably translated into proteins. As
a result, current R&D efforts in the mRNA
field focus on improving stability, duration of
expression, delivery efficiency, and target
mRNA Translation Termination
specificity
to
expand
its
therapeutic
applications.
mRNA Stability
Figure 1 The Function of poly(A) Tail in mRNA
Translation and Stability.
Why Poly(A) Control is important for
mRNA?
The poly(A) tail, comprising a string of
adenine nucleotides at the 3’ end of most
eukaryotic mRNAs, plays a critical role in
maintaining mRNA stability and facilitating
efficient translation. In eukaryotic cells, the
poly(A)-binding protein (PABP) binds to the
poly(A) tail and interacts with the mRNA's 5’end
to form a closed-loop structure. This structure
protects the 5’ cap from hydrolysis and prevents
deadenylation and degradation, thus promoting
mRNA stability in vivo [2] (Figure 1).
Currently, most manufacturers prefer the
direct transcription from a poly(A)-containing
template plasmid rather than enzymatic post-
transcriptional tailing with poly(A) polymerase.
This approach demands robust control strategies
to ensure consistent poly(A) length and purity
throughout the entire process, from upstream
plasmid synthesis to downstream mRNA
production.
Theoretically, PABP typically binds the
poly(A) tail with the length of mRNA designed at
least 30A. In the mRNA therapeutics industry,
tails are typically designed to be ~100 adenines
in length, which will be determined and verified
at early priority during drug design and discovery.
2,000,000
1,600,000
1,200,000
800,000
400,000
0
Poly(A) Length of mRNA
Figure 2 Expression Activity of mRNA with
Different Length of poly(A) Tail.
mRNA Translation Activation
2
ProBio’s Solutions to Meet Challenges of Poly(A) Control During Plasmid and mRNA
Manufacturing
PROCESS
Sequence Design
CHALLENGES
STRATEGIES
Inappropriate sequence design
Preliminary risk analysis and sequence optimization before manufacturing
Host strain selection: long poly(A) tail is unstable during plasmid
replication
Passaging stability: poly(A) purity decreases with the increase of passage
generation
Plasmids with longer poly(A) tend to be polymer supercoiled
Poly(A) stable strain is recommended
Well-established poly(A) IPC system
Cell Banking
Template
Plasmid
Various processes available
Complete production process control strategy
ProSyn cell-free process without fermentation
Poly(A) tracts shortening occurred during plasmid replication in e.coli
Manufacturing
T7 RNA polymerase slippage in vitro transcription.
Purification Based on oligo dT is hard to separate short and long
poly(A) tailed mRNA
Comprehensive and diversified biosynthesis and purification platform
Professional and experienced execution and delivery in mRNA CMC
projects
mRNA
Manufacturing
Sanger Sequencing for poly(A) analysis is susceptible to affected by
polymerase slippage
mRNA is sensitive to pretreatment condition
Analytical
Control
Orthogonal analytical tools for monitoring poly(A) purity
Challenges and Solutions of Poly(A) Control
in Template Plasmid Manufacturing
Ensuring the integrity and stability of the
poly(A) sequence during template plasmid
manufacturing is the key to achieve high-quality
mRNA products. To address this, ProBio has
implemented comprehensive control strategies
covering plasmid backbone design, strain
selection, cell banking, and fermentation
processes.
Figure 3 Plasmid poly(A) region sanger
sequencing analysis (at length A120). Less
impure peaks (B) using ProBio proprietary
IntegraBone, compared to (A) using commercial
template plasmid backbone.
Sequence Design
Critical elements such as the origin of
replication, selection markers, and gene insertion
sites in plasmid vectors can impact poly(A)
sequence stability [9]. In order to guarantee the
poly(A) stability, ProBio has developed a
universal mRNA plasmid backbone, IntegraBone,
optimized to maintain poly(A) tail integrity for
lengths up to 150 adenines (a 120 A comparison
is shown in Figure 3).
Cell Banking
Beyond backbone design, ProBio has
engineered a proprietary poly(A) stable strain,
PowerS™-PolyAssure, specifically for plasmids
with long poly(A) tails. PowerS™-PolyAssure
3
strain significantly enhances poly(A) sequence
stability during cell propagation. Compared to
commercial strains, ProBio's proprietary PowerS
™-PolyAssure strain achieves a 100–500%
higher rate of positive clones in cell banking
(Figure 4) and demonstrates superior passaging
stability (Figure 5), resulting in higher-quality
plasmid production.
Analytical Control
ProBio employs advanced analytical
technologies to monitor poly(A) tail purity as a
critical quality attribute throughout production.
While Sanger sequencing is commonly used, it
is prone to polymerase slippage in homopolymer
regions. ProBio has therefore developed a high-
resolution capillary gel electrophoresis (CGE)
method that enables precise, reproducible
assessment of poly(A) purity with 2–5 base pair
resolution, <4% RSD, and 90–110% accuracy
(Figure 6).
Positive Clone Rate
100
80
60
40
20
0
Poly(A) Purity During Plasmid
Manufacturing Process
105
100
95
A
B
Plasmid
C
90
85
80
75
Commercial Strain
ProBio Proprietary Poly(A) Strain
70
Figure 4 Comparison of the Positive Clone Rate
by Different Strains
Project A
Project B
Project C
Figure 5 Comparison of Plasmid Poly(A)
Stability (at length A120) in Different Strains
Figure 6 Poly(A) purity in plasmid production
process for different molecules
(A) Growth Performance
Fermentation and Process Optimization
Drawing on its extensive experience in
process development, ProBio has optimized
fermentation parameters for plasmids with
poly(A) sequences. These improvements have
led to a 300–600% increase in plasmid yield and
a 50–150% increase in OD600, all while
maintaining poly(A) sequence integrity (Figures
6 and 7).
250
200
150
100
50
0
A
B
C
Plasmid
Before After
4
improving
the
resolution
for such
of
affinity
(B) Fermentation Yield
chromatography
populations.
1200
1000
800
600
400
200
0
Additionally, a non-affinity purification method
based on hydrophilicity principles has been
successfully implemented, further increasing
poly(A) purity during mRNA production (Figure
5).
A
B
C
100
Plasmid
Before After
90
Figure 7 Optimization results of fermentation
process for low-copy plasmid molecules
80
Challenges and Solutions of Poly(A) Control
During mRNA Manufacturing
70
Traditional Process Process-1
Process-2
Poly(A) purity is also influenced during
mRNA synthesis, where T7 RNApolymerase can
Figure 5 Chromatography Process Optimization
Improves mRNA Purity
slip
in
homopolymeric
adenine
regions,
producing heterogeneous poly(A) tail length [4].
Addressing this requires both upstream and
downstream process optimization.
Poly(A) Purity (HPLC): 93.6%
Poly(A) Distribution (LC-MS): 98-105A
mRNA Process Optimization
101A
99A
100A
102A
103A
ProBio has established a mRNA process
toolbox designed to enhance T7 polymerase
efficiency and reduce slippage during in vitro
transcription (IVT). This enables higher poly(A)
purity post-IVT and supports mRNA yields up to
14 g/L. Downstream process enhancements—
such as tailored chromatography method, buffer
system, and flow rate—further contribute to
consistent poly(A) tail profiles, reaching poly(A)
purity up to 90%.
98A
104A
105A
Figure 6 Characterization of mRNA poly(A)
Tail using LC-MS
Orthogonal Analytical Tools for Monitoring
Poly(A) Purity
Aligned with regulatory guidance from the
WHO, NMPA, and USP [4–7], ProBio employs
a suite of orthogonal analytical methods to
assess poly(A) length and distribution in both
plasmids and mRNA.
Chromatography Strategies
Traditional affinity chromatography uses
Oligo(dT) ligands to bind the poly(A) tail,
enabling efficient isolation of polyadenylated
mRNA. However, this method has limited
resolution for separating mRNAs with different
poly(A) tail lengths. ProBio is experienced in
For example:
•
LC-MS (Liquid Chromatography-Mass
Spectrometry): analyze distribution and
percentage of the poly(A) tail at single
nucleotide resolution
5
•
IP-RP-HPLC (Ion-Pair Reversed-Phase
HPLC): separate different lengths and
percentage of varied poly(A) tail
e66493.
[4] Macdonald L E, Zhou Y, McAllister W T.
Termination and slippage by bacteriophage T7
RNA polymerase[J]. Journal of molecular
These methods enable precise poly(A)
characterization, providing a strong foundation
for consistent mRNA product quality and
regulatory compliance.
biology, 1993, 232(4): 1030-1047.
[5] Evaluation of the quality, safety and
efficacy of messenger RNA vaccines for the
prevention of infectious diseases: regulatory
considerations, World Health Organization,
2021.
ProBio CDMO Service
ProBio is a cutting-edge Contract
Development and Manufacturing Organization
(CDMO) committed to providing end-to-end
support across the mRNA therapeutic value
chain—from plasmid production and mRNA
synthesis to drug product manufacturing.
Our integrated CMC development services
are designed to accelerate the development of
innovative mRNA therapeutics, empowering our
partners for better serving worldwide.
[6] Technical Guideline for the Pharmaceutical
Study and Evaluation of In-vivo Gene Therapy
Products (Interim), Center for Drug Evaluation,
2022.
[7] Guideline for the Development of
(Cellular) Immunotherapy Products Based on
mRNA-LNP Technology, China Food and
Drug Corporation Quality and Safety
Promotion Association, 2024.
[8] Analytical Procedures for Quality of
mRNA Vaccines and Therapeutics (Draft
Guideline, 3rd Version), USP’s BIO3 Expert
Committee, 2024.
References
[1] Biziaev N, Shuvalov A, Salman A, et al.
The impact of mRNA poly (A) tail length on
eukaryotic translation stages[J]. Nucleic Acids
Research, 2024: gkae510.
[9] Lukić I, Dragačević L, Panić M, et al. mRNA
vaccine manufacturing–challenges in plasmid
[2] Passmore L A, Coller J. Roles of mRNA
poly (A) tails in regulation of eukaryotic gene
expression[J]. Nature Reviews Molecular Cell
Biology, 2022, 23(2): 93-106.
DNA
cloning
vector
design[J].
Microbiology/Mikrobiologija, 2024, 45(1): 3-9.
[10] Dhir A, Winckler C, Mañas C, et al.
Synthetic, enzymatically produced dna for gene
therapy and vaccine applications[J]. Cytotherapy,
2024, 26(6): S133-S134.
[3] Xiang K, Bartel D P. The molecular basis
of coupling between poly (A)-tail length and
translational efficiency[J]. Elife, 2021, 10: