Innovations in AAV Production: Leveraging High-Yield Cell Lines, novel RC plasmid, and Dual-Plasmid Systems

Abstract

In the dynamic landscape of gene therapy, PackGene has pioneered innovative approaches to meet the escalating demand for high-quality adeno-associated virus (AAV) vectors. Through the proprietary π-Alpha 293 AAV High-Yield Platform, PackGene achieves remarkable enhancements, with a 3 to 8-fold increase in AAV production yield across various serotypes. The journey begins with the development of the PCS3.0 suspension cell line, exhibiting substantial improvements in production yield compared to its predecessors. Utilizing cutting-edge technologies, PackGene meticulously engineers PCS3.0 for optimal performance, resulting in unparalleled productivity, stability, and quality. Process optimization, coupled with the introduction of a novel dual-plasmid system, further elevates AAV productivity and quality while minimizing the risk of contamination. In conclusion, PackGene’s commitment to innovation revolutionizes AAV production, driving transformative advancements in gene therapy.

Introduction

In the rapidly evolving landscape of gene therapy, the demand for high-quality adeno-associated virus (AAV) vectors continues to soar. To meet this demand and advance the field, PackGene has pioneered innovative approaches to enhance AAV production, focusing primarily on the development of a high-yield monoclonal AAV production cell line, modified RC plasmid, and a novel dual-plasmid system.

π-Alpha 293 AAV High-Yield Platform

PackGene’s proprietary π-Alpha 293 AAV High-yield Platform uses uniquely designed RC plasmid in the triple-plasmid transfection system to increase AAV production by 3 to 8 times for various AAV serotypes. This technology is paired with both in-process upstream and downstream QbD optimizations to increase total AAV yield up to 10 fold. A single batch of AAV production delivers up to 1E+17vg virus particles, which is enough to meet the needs of most clinical and commercial level of AAV production.

Advantages of the Platform

• Productivity: Proprietary technologies elevate AAV yield in HEK293 serum-free cell suspension systems by over 10-fold.
• Quality: Unique process development procedures significantly diminish key impurities such as Host Cell DNA (HCD) and endotoxin.
• Viability: Key technological innovations reduce the rate of empty capsids and enhance infection titers.

High-Yield AAV Production Cell Line: From PCS 2.0 to PCS 3.0

PackGene’s journey towards high-yield AAV production commences with the development of a single clonal suspension cell line. Derived from the ATCC HEK293 cells, PackGene’s monoclonal cell line yields impressive results. With Production yield enhancements of PCA (adherent) up to 7x titer and PCS 2.0 (suspension) up to 4x titer, the progression to PCS 3.0 represents a significant leap forward.

After obtaining the PCS2.0 monoclonal cell line, we proceeded to evaluate multiple monoclonal cell lines to identify a superior candidate, resulting in the development of the PCS3.0 line. Acknowledging the crucial role of cell substrates in AAV manufacturing, PackGene’s researchers meticulously optimized PCS3.0 for peak performance. Leveraging state-of-the-art technologies such as Dispencell in-situ cell dispensing and CloneSelect Imager for clonality verification, we generated highly uniform monoclonal cells. These cells underwent rigorous screening based on AAV production yield and Population Doubling Time (PDT), leading to the identification of the most efficient clone. The selected PCS3.0 cell line underwent further refinement through gradient-decreasing serum cultures and two rounds of single-cell sorting (Figure 2). This culmination of efforts resulted in the establishment of a robust suspension cell line boasting unparalleled AAV productivity, stability, and quality.

𝝅-Alpha 293 High-yield AAV Production Platform Process Optimization

Recovery rate and scalability without compromising quality:

With an overall recovery rate exceeding 30%, our suspended production process demonstrates robust amplification across varying scales, from SF125 to ambr250/3L/50L/200L, while maintaining quality. Successful 200L scale-up AAV production, stable genomic titer, and residual hcDNA during scale-up have resulted in 1.0E17 vg at harvest, meeting stringent GMP production requirements for diverse AAV serotypes in downstream processes. Key Product Quality Attributes, including significant full capsid enrichment, are maintained at similar levels during scale-up from 76-92%.

Process Optimization of CPP Increases Total AAV Yields by 10 times:

Process optimization of Critical Process Parameters (CPP) has resulted in a remarkable tenfold increase in total AAV yields. This optimization encompasses various facets including transfection conditions such as cell density, total plasmid amount, plasmid ratio, and DNA/PEI ratio. Engineering parameters such as stir speed, pH, dissolved oxygen (dO2), and temperature have also been meticulously fine-tuned. Additionally, lysis and harvest conditions including stir speed, density, time, and temperature have been optimized to ensure maximal AAV production efficiency (Figure 4).

Uniquely Designed RC Plasmid Increases AAV Yield by 3-8 Times

A Novel Rep-Cap plasmid containing a proprietary non-coding regulatory element enhances AAV yield in both adherent and suspension cell lines by 3-8 times in various serotypes (Figure 5 A-B). By employing novel plasmid design and optimization, the encapsidated plasmid DNA ratio decreases from 3% to 0.1%~0.2%. (Data not shown)

Novel Dual-Plasmid System: Next-Generation AAV Production Strategy

The traditional triple-plasmid transfection system, a cornerstone in industrial AAV manufacturing due to its scalability, encountered significant hurdles. Approximately two decades ago, a dual-plasmid system emerged as a cost-saving and simplification measure. Despite its adoption, persistent challenges including low productivity, quality issues, and the risk of replication-competent AAV (rcAAV) necessitated further innovation. Our study presents a novel dual-plasmid system featuring a uniquely engineered TP vector, demonstrating superior AAV productivity and quality compared to the traditional triple-plasmid system, with minimal rcAAV risk.

Process development entailed screening seven transfection reagents to identify a cost-effective option for high-titer production. Optimized parameters were implemented to enhance productivity and mitigate impurities. Scalability assessments conducted on Ambr 250 and 3L stirred bioreactors revealed equal or higher yields with reduced impurities.

Concurrently, PackGene pursued innovative strategies to optimize AAV production processes alongside cell line development. The introduction of the dual-plasmid system marks a significant milestone in AAV production technology. By consolidating RepCap and adenovirus helper genes into a single plasmid, this system offers heightened productivity and quality while minimizing the risk of rcAAV contamination.

Central to the success of the dual-plasmid system is the design of the TP vector, which features optimized sequences and arrangement of vector elements. Through meticulous experimentation and optimization of transfection reagents and process parameters, PackGene achieved exceptional AAV yields while concurrently reducing process-related impurities. Our initial design, TP007 plasmid, encompassed p5, Rep & Cap, and E2A&E4&VA RNA elements, culminating in successful packaging.

Non-rcAAV design of packing plasmids for dual-plasmid system.

We have developed a non-replication-competent adeno-associated virus (rcAAV) design for packing plasmids within our dual-plasmid system. Unlike the tri-plasmid system, which includes separate plasmids for the gene of interest (GOI), replication-competent (RC) genes, and helper functions, our dual-plasmid system comprises a TP plasmid and a GOI plasmid. This innovative approach enhances AAV productivity and quality compared to the traditional triple-plasmid system while minimizing the risk of rcAAV contamination. As shown in Figure #, TP011 is a well-designed dual-plasmid vector with insertion of a unique non-coding regulatory element (RE). Compared to traditional TP007 design, TP011 system shows higher AAV productivity and minimizes the risk of rcAAV.

DoE optimization enabling high productivity, further increase 3x

Through Design of Experiments (DoE) optimization, we have unlocked the potential for significantly higher productivity, resulting in a remarkable threefold increase. This systematic approach allows us to explore and optimize various parameters simultaneously, leading to enhanced AAV production efficiency. By meticulously fine-tuning critical process parameters such as cell density, total plasmid amount, plasmid ratio, DNA/PEI ratio, as well as engineering parameters including stir speed, pH, dissolved oxygen (dO2), and temperature, we have achieved unparalleled levels of productivity. This optimization not only maximizes AAV yield but also ensures consistent and high-quality vector production, further advancing the efficacy and reliability of our AAV production platform.

We also screened for optimal transfection reagent (TR) for dual-plasmid system (Figure 7A) and identified a small molecule compound significantly enhanced AAV9 productivity on different TRs (Figure 7B).

TP011-dual system with higher yield and lower HCD residue.

Our TP011-dual system represents an advancement in AAV production technology, shows superior yield and reduced host cell DNA (HCD) residue. The TP011 vector, meticulously engineered with optimized sequences and arrangement of vector elements, facilitates enhanced AAV production efficiency. The TP011-dual system streamlines the production process, resulting in heightened yield and purity. Furthermore, rigorous quality control measures ensure that HCD residue levels are kept to a minimum, maintaining the integrity and safety of the produced AAV vectors. This remarkable combination of increased yield and reduced impurities positions the TP011-dual system as a leading solution in the field of AAV vector production, offering researchers and clinicians a powerful tool for advancing gene therapy applications. Based on optimized transfection process and two promising TRs, TP011 dual-system shows higher lysate titer and lower residual host cell DNA (HCD) impurities than triple-system. Notably, there is

Application of dual plasmid system in therapeutic case studies

The TP011-dual system demonstrates its versatility in accommodating therapeutic Gene of Interest (GOI) across various AAV serotypes, showcasing high productivity and efficacy. In a gene therapy scenario utilizing scAAV9, the dual-plasmid system significantly reduces residual plasmid DNA, ensuring enhanced safety and efficacy of the therapeutic intervention.

Dual-TP011 system shows higher AAV productivity on different therapeutic GOIs and serotypes including AAV2, AAV5, AAV8 and AAV9; We also developed a novel dual vector TP013 with truncated Ad helper genes, which shows higher lysate genome titer on a ssAAV9-GOI-1 project, approximately 8 folds yield compared to tiple system.

Conclusion:

PackGene’s unwavering dedication to innovation and excellence has propelled the advancement of high-yield AAV production platforms, ushering in a new era in gene therapy. The development of the PCS3.0 cell line and our novel dual-plasmid system epitomize PackGene’s commitment to pushing the boundaries of AAV manufacturing processes, equipping researchers and clinicians with potent tools to expedite the development of groundbreaking therapeutics.

As the field of gene therapy continues to evolve, PackGene remains at the forefront, driving innovation and shaping the future of AAV production. With an unwavering commitment to excellence, PackGene is spearheading transformative advancements in gene therapy, paving the way for revolutionary medical breakthroughs.

For deeper insights into PackGene’s cutting-edge AAV production technologies, we encourage you to explore our video and visit our website. Immerse yourself in the future of plasmid design and AAV production with PackGene, where innovation knows no bounds.

PackGene Biotech is a world-leading CRO and CDMO, excelling in AAV vectors, mRNA, plasmid DNA, and lentiviral vector solutions. Our comprehensive offerings span from vector design and construction to AAV, lentivirus, and mRNA services. With a sharp focus on early-stage drug discovery, preclinical development, and cell and gene therapy trials, we deliver cost-effective, dependable, and scalable production solutions. Leveraging our groundbreaking π-alpha 293 AAV high-yield platform, we amplify AAV production by up to 10-fold, yielding up to 1e+17vg per batch to meet diverse commercial and clinical project needs. Moreover, our tailored mRNA and LNP products and services cater to every stage of drug and vaccine development, from research to GMP production, providing a seamless, end-to-end solution.

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