Poster

PowerS™ - ITRrs Strain Suitable for Different AAV Transfer Plasmids

Proprietary PowerS™-ITR Strain Suitable for Different AAV

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

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 ,

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:

Cell Bank

Process

Plasmid

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.

ProBioCDMO.com