Poster
PowerS™ - ITRrs Strain Suitable for Different AAV Transfer Plasmids
Proprietary PowerS™-ITR Strain Suitable for Different AAV
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Transfer Plasmids
Background
Agarose gel electrophoresis (AGE) is utilized to distinguish DNA fragments
Higher Plasmid Yield (Up to 1 g/L)
of varying molecular sizes and configurations based on their differential
migration rates in an electric field. It has been observed that the presence
of CG-tails just outside the ITR region can introduce additional instability.
This issue can be effectively addressed by removing these sequences
using specific restriction enzymes at cutting sites (refer to Fig.1).
Subsequently, the rate of ITR recombination can be precisely charac-
terized through enzyme digestion followed by AGE grayscale analysis
(refer to Fig. 4).
Cell Bank Construction
Adeno-associated virus (AAV) is increasingly recognized as a
powerful tool for gene transfer and genome editing applications in
gene therapy. Its effectiveness hinges on two short inverted terminal
repeats (ITRs) flanking the AAV genome. These ITRs contain essential
cis-acting elements necessary for the rescue, replication, and
packaging of the genome (refer to Fig. 1). However, the structural in-
stability of ITRs often leads to deletions during the propagation of
AAV transfer plasmids. This results in reduced plasmid yields, lower
The well-established and characterized clone ensures excellent ITR integrity
and genetic stability
Comprehensive documentation for traceability and repro ducibility
Compliant to regulatory standards and guidelines; rigorous testing for
contaminants (bacteria, fungi, mycoplasma)
viral titers, decreased packaging efficiency, and increased variability in
downstream process. Addressing these challenges is crucial;
therefore, developing a stable host strain and optimizing the
production process for manufacturing AAV transfer plasmids is
imperative. Our focus on enhancing the stability of ITRs and refining
manufacturing techniques ensures high-quality, efficient production
of AAV vectors, paving the way for reliable and groundbreaking
advancements in gene therapy.
Process Development
Sanger Sequencing
Extensive expertise in process development with thorough understanding of
the product and its intended use. Utilize Quality by Design (QbD) to ensure
product quality and process for maximal efficiency. Develop and integrate
in-process control strategy to ensure the process to remain within defined
tolerances.
Fig.3 Fermentation yield of AAV transfer plasmids.
Stable Passage Stability (≥ P10)
Table.1 Passage stability of AAV transfer plasmids.
Manufacture and Release
ProBio offering: GMPro™ and GMP Grade plasmid DNA manufacturing
Experience with navigating regulatory pathways in multiple regions, including
FDA, EMA and NMPA
Complaint to Good Manufacturing Practice (GMP) guideline
Validation and qualification of equipment and processes
Comprehensive documentation of all manufacturing activities, decisions,
and validation efforts
Fig.5 Sanger sequencing analysis. Less impure peaks (B) denote lower ITR
mutation and deletion rate, compared to (A).
Restriction mapping, while effective for analyzing ITRs, does not detect
variants characterized by small deletions that do not affect restriction
recognition sequences. For a more comprehensive analysis of plasmid
sequences, including the integrity of ITR regions, the double deoxy chain
termination method, pioneered by Frederick Sanger, is employed extensively
(refer to Fig. 5).
Fig.1 The secondary structure of the ITR. The characteristic 'T' shape of the ITR
is formed by two adjacent inverted repeats (BB' and CC') separated by a single
unpaired nucleotide.
Analytical Methodology of ITRs
The digestion of ITR regions, critical for gene therapy applications, primarily
employs two methodologies: restriction enzyme analysis and Sanger
sequencing. These techniques provide comprehensive insights into the
structural integrity and sequence fidelity of ITR regions, essential for effective
gene therapy vector development.
PowerS™-ITR Strain Performance
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ProBio has dedicated extensive research to addressing the critical
challenges in AAV plasmid production. Our efforts have culminated in
Plasmid Manufacture
Conclusion
the development of a proprietary recombinant strain, PowerS™-ITR ,
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specifically engineered to effectively resolve issues related to ITR re-
combination. This innovative solution enhances the stability and effi-
ciency of AAV production, setting a new standard in the field.
Restriction Enzyme Digestion
ProBio offers the proprietary PowerS™-ITR strain, characterized by:
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Cell Bank
Process
Plasmid
01
02
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Clear-sourced, detailed documentation for traceability and reproducibility
Excellent performance of ITR recombination control (<10%) and well-pre-
served ITR regions
Construction
Development
Manufacture
Excellent ITR Integrity (>90%)
Up to 1 g/L plasmid yield in bioreactor
Exceptional genetic stability (≥P10)
Compatibility with various client-specific AAV plasmids
• Primary Cell Bank, PCB
• Master Cell Bank, MCB
• Working Cell Bank, WCB
• USP Process Dev.
• DSP Process Dev.
• Process Validation
• GMP Plasmid Manufacture
• QC testing & Release
This strain is genetically modified and developed to enhance the efficiency
and reliability of AAV transfer plasmid production with excellent performance
on ITR integrity and yield under a cost-effective and efficient process.
Fig.6 Three stages of plasmid manufacturing process.
Quality analysis and quality control of ITR regions are rigorously implemented
at every stage of the manufacturing process.
Fig.4 AGE analysis of restriction enzyme digestion. During enzyme digestion, (A) and
(B) show a missing Ahd I (BB') and Sma I (CC) site respectively, signifying ITR deletion.
Fig.2 ITR recombination rate of AAV transfer plasmids.
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