Proprietary Cell-Line Development for High-Titer AAV Manufacturing

Published on: 
BioPharm International, BioPharm International, March 2024, Volume 37, Issue 3
Pages: 10-15

Proprietary cell lines offer opportunities for achieving high AAV titers.

The growing demand for adeno-associated viral (AAV) vectors in therapeutic applications emphasizes the need for scalable and more efficient manufacturing methods. Establishing proprietary cell lines offers substantial opportunities for achieving high AAV titers. High titers allow shorter production times, rapid production cycles, higher throughput, simplified downstream processes, less likelihood of contamination, and ultimately can lead to greater flexibility in process scale-up, all of which can contribute to a reduction of costs and product variability, according to Ines do Carmo Goncalves, head of project management for cell-line and process development at Cytiva.

Proprietary cell lines may also lower the cost of input materials, improve cell growth properties, and/or improve the quality of the vector product. As such, it is possible for every AAV manufacturing process, regardless of the type of cell, to benefit from cell-line development, according to James Cody, associate director of technical sales and evaluations at Charles River.

Several approaches can be taken: isolation of high-producing natural clones for transient transfection or infection processes; engineering of cells for the same processes; and development of packaging/producer cell lines that eliminate the need for transfection/infection steps. The optimal technique depends on the nature of the AAV vector and intended therapeutic application.

Advantages of clonal selection

The most widely used approach to cell-line development for AAV manufacturing is single-cell isolation and cloning. “This method offers the capacity to screen thousands of clones, allowing for the identification and isolation of highly productive and robust cell lines for further process development,” says Goncalves.

Notably, incorporating digital software for data management in the process enhances the ability to detect and identify genetic characteristics that were previously unknown, Goncalves adds. “Identifying these traits can help improve outcomes, such as increasing AAV titers or the percentage of full capsids. Once well characterized, these traits can then be introduced into other cell lines,” she comments.

Upon isolation, single cells are cultivated into clonal populations, ensuring genetic uniformity within the selected clone and contributing to the stability and consistency of the cell line, according to Silvia Ungari, upstream vector development manager for AGC Biologics.

Ideally, Ungari observes, selection and cloning are coupled with some type of imaging technology to provide documentation of single-cell sorting and growth, which allows for demonstration of final monoclonality in a single cloning run. Fluorescence-activated cell sorting (FACS) is an advanced method that enables the isolation of individual cells based on specific fluorescence markers that correlate in some way to higher vector titers, providing a means to select cells with particular characteristics, she notes.

SK pharmteco, according to Brian Tomkowicz, senior director of research and development, uses cell printing with high content imaging for selection and cloning. “With this efficient process, it is possible to plate one 96-well plate of single-cell clones in about four minutes. It is, in addition, a completely closed system that does not require a trained operator, as is necessary when using a flow cytometer capable of sorting,” he says.

One of the key advantages of single-cell selection and cloning is avoidance of the need for genetic engineering. “The optimization of a cell line for AAV production via the selection of clones from a bulk population with improved viral protein expression, or packaging efficiency, or other positive mechanisms resulting in an overall increased productivity, allows the identification of naturally occurring high-performing clones without genetic manipulation. These cells are more readily approved by regulatory agencies, potentially expediting the approval process for AAV production,” Ungari explains.

Consistency due to the uniform behavior of a specific clone rather than a heterogeneous bulk population is another advantage, according to Ungari. “Proprietary cell lines typically ensure batch-to-batch reproducibility and reduce variability in AAV titers during large-scale manufacturing,” she says.

To realize optimum results in cell-line development, Ahmed Youssef, senior manager of upstream process development at Ascend, emphasizes the importance of focusing on functional AAV titer as the primary read out. “High vector potency is key to improving both the quality and safety and reducing the cost of goods of a given product,” he comments.

Genetic engineering

Genetic engineering is used to add desired and/or remove undesired traits from cell lines for production of AAV vectors. As such, genetic modifications can enable not only increased titers, but increased AAV particle quality, according to Goncalves.

It is also possible, observes Bingnan Gu, head of viral-vector R&D with Lonza, to develop novel host-cell lines for AAV production from scratch. “It is essential with this approach to have full documentation of the process and materials used to achieve cell-line establishment/immortalization and to meet safety requirements, such as the absence of any adventitious agents.”

For a given host AAV cell line, one can either perform screening to identify clonal cell lines that can support relatively higher AAV productivity (compared to parental cells), or carry out rational cell-line engineering to upregulate or downregulate host-cell gene expression or introduce new ‘helper’ genes that could lead to increased AAV productivity, Gu explains.

The use of genetic modifications should, Ungari comments, be approached cautiously due to regulatory considerations.

Packaging/producer cell development

In a packaging cell line, the helper genes and Rep-Cap genes are stably integrated into the cell genome and the AAV is produced upon induction of these genes and transfection of the gene of interest (GOI). In a complete AAV stable producer cell line (PCL), not only the helper and Rep-Cap genes but also the GOI are stably integrated in the cell. AAV production with stable cell lines is triggered upon induction of these viral genes without any helper virus infection or plasmid transfection. An AAV PCL, according to Gu, can significantly improve AAV titer and quality and enable robust large-scale production beyond 2000 L at much reduced cost and complexity.

Cost reduction is achieved because packaging/producer cell lines eliminate the need for costly plasmid scale-up and transfection reagents, according to Tomkowicz. Yields may also be improved without having to necessarily increase scale, he adds, as packaging/producer lines allow the end user to intensify production processes by taking advantage of high density in combination with perfusion culture.

“The key to successful development of packaging/producer cell lines is genetic manipulation of cellular machinery to mitigate host cellular immune responses against the AAV product, which to date has been investigated using a variety of approaches,” Tomkowicz says.

The challenge then becomes the time it takes to establish stable producer cell lines. “As a result, this approach to cell-line development is highly recommended for programs requiring high amounts of recombinant AAV (rAAV), such as preclinical studies performed in large animals, or gene therapies requiring large doses or involving large patient populations (e.g., cardiovascular diseases or diabetes),”Tomkowicz contends.

Both Youssef and Ungari agree that producer cell lines are more suitable for large-scale production and commercialization. “Stable cell lines prove to be better suited for advanced stages of clinical trials or commercial production, particularly due to the considerable time investment needed for their establishment and the requirement for a finalized process. An advantage is that in these phases, higher volumes of vector may be needed, which can be more easily produced in a stable manner,” Ungari notes. Packaging cell lines, meanwhile, are primarily used for small-scale rAAV production in research settings, according to Youssef.

Indeed, Cody believes that packaging and producer cell lines fit within a continuum of production cell options. “The choice of which cell line is best may come down to the timeline to the clinic and the overall goals of the program. A true producer cell line may take the longest time to develop, but may be the most cost-effective approach for commercial production of a gene therapy product,” he observes.

Proprietary packaging and producer cell lines play a crucial role in the advancement of the AAV gene therapy field, according to Goncalves. “They enable the production of high titers of AAV vectors with high quality while also ensuring scalability and reproducibility. In addition, the use of stable packaging and producer cell lines offers a massive reduction in the costs of goods for producing AAV vectors in quantities that meet the industry demand,” she states.

It is that cost reduction potential of producer cell lines that most interests Marguerite Campbell, associate director of research and development at SK pharmteco. “Million-dollar gene therapies are not sustainable for rare-disease individuals or large patient populations suffering from more prevalent diseases. Having off-the-shelf packaging lines with easy conversion to producer cells not only democratizes gene therapies, but provides benefit where it is needed most,” she comments.

The type of cell matters

While all cell types can benefit from cell-line development efforts such as enhancing packaging capacity or ensuring robust expression of AAV components, the specific actions required may differ based on the manufacturing process mechanisms involved.

Insect cell lines, for instance, suffer from heterogenous AAV vector packaging and thus require specific attention to vector integrity, says Youssef. Cell-line development for these cells also tends to focus on facilitating efficient viral entry or evading the anti-viral immune response, according to Ungari. Mammalian, and more specifically human cell lines, used for transient transfection processes, meanwhile, present process intensification challenges, Youssef notes. As a result, proprietary cell lines often are customized for improved transfection efficiency, Ungari remarks. “It is therefore crucial to tailor the optimization of each cell line according to its specific manufacturing process and specific product,” she concludes.


When it comes to developing packaging/producer cell lines, Tomkowicz notes that while transitioning from a triple transfection-based process is relatively straightforward because downstream considerations for purification and analytics are comparable, each cell line (HEK293, Sf9, etc.) presents its own challenges, and the characteristics of the selected cell line (origin/derivation, doubling time, and permissiveness for viral infection and replication) determine the efficiency of viral productivity. He adds that because the insect platform is completely distinct from mammalian HEK293 systems, it often requires more cell and viral engineers/scientists knowledgeable in the design and execution of the two separate platforms.

Hurdles to overcome

Developing proprietary cell lines can be a complex and challenging process. The main obstacles lie in the complexities of cellular biology, Goncalves notes. “Guaranteeing high productivity involves complex factors such as packaging efficiency and/or viral genome replication. Achieving maximized productivity demands a deep understanding of how to balance these factors. This level of optimization requires extensive genetic characterization and process development activities,” she observes.

Campbell highlights the need to have a validated cell line to start with as a barrier to beginning cell-line development. “While commercial cell lines exist, further engineering to include pHelper and/or Rep/Cap constructs is strictly prohibited [by regulators]. In addition, finding scientists who can engineer viral packaging constructs and who know how to single-clone and expand cells to GMP [good manufacturing practice]-banking is currently a challenge, as can be gaining access to advanced high-throughput technologies for accelerating cell isolation, screening, and selection. Once those issues are overcome, there is still a need to optimize cell-culture processes and customize the cell-culture media,” she explains.

Screening of cell lines, including selecting the best criteria for screening, is a significant challenge noted by Cody. In addition, he notes that cell lines can take significant amounts of time to develop, screen, and characterize, so unless the increased productivity is robust, it may not always be possible to recoup the cost of cell-line development. The need for comprehensive characterization of new cell lines can also be a hurdle. “Depending on the origin of the cell line and how it was generated, new characterization assays may need to be developed and will be dictated by the type of platform,” he says.

Other technical challenges pointed out by Tomkowicz include the need to assess anti-AAV immunity for understanding clinical data and product development, the need for a rapid assay to quantify infectious viruses and gene therapy vectors, and efficient harvest of AAV vectors from cell-culture media.

Furthermore, according to Youssef, cells affording high titers may not be easy to grow or maintain in culture; prevention of AAV particle degradation via proteases, nucleases, and shear forces is necessary by ensuring high cell viability despite harmful gene products expressed and required for AAV manufacturing; and the safety of AAV particles must be assured through testing for potential contaminants, pathogens, and host-cell genes.

For development of producer cell lines affording high AAV titers, the main challenge also gets back to biological mechanisms. “There is an incomplete understanding of the molecular mechanism that controls AAV production in host cells,” comments Gu. He adds that the necessity to demonstrate cell-line stability for any new cell line regardless of its derivation, which involves several months of testing, represents another impediment.

All these challenges, concludes Ungari, “underscore the need for a comprehensive approach, integrating growth efficiency, viability, stress-related cell resistance, aggregation profile, packaging efficiency, material costs, genetic stability, and regulatory compliance for the successful creation of high-titer AAV-producing proprietary cell lines.”

Trade-offs to manage

Ideally, observes Cody, an ideal cell line will improve not just overall productivity, but also product quality (lower residuals, higher percent-full capsids, better infectivity, etc.). Cell-line development, however, brings with it a number of new considerations, many of which play off one another, requiring the management of the benefits and risks. These trade-offs, Youssef says, can be complex and vary depending on the specific manufacturing platform and product being developed.

For one thing, introducing a new cell line into an existing process necessitates a revision of the timing and steps in the process and may impact downstream processing requirements, raw-material consumption and costs, and full/empty capsid ratios, which can potentially affect the overall potency, safety, and therapeutic effectiveness of the AAV product, according to Ungari. There can also be a threshold beyond which higher titers do not afford proportional therapeutics effects.

Changes in productivity can also change the density of cells needed during production, which could in turn impact residuals such as host-cell proteins and/or host-cell DNA (for better or for worse), adds Cody. “It is best to base tradeoff decisions on data, which is why it is important to test the critical quality attributes (CQAs) of AAV vectors made from a new cell line at a scale representative of GMP production,” he remarks.

Potency can also potentially be reduced in high-titer AAV products due to increased aggregation, which can also increase the risk of side effects, according to Youssef. The introduction of new genetic sequences may also necessitate additional release testing.

“Aiming for higher AAV titers in the production process inevitably involves finding a delicate balance with other crucial factors. Striking the right balance between titers and potency, in particular, becomes essential in the optimization process,” Ungari contends. That requires a comprehensive understanding of the process and product and application of a quality-by-design approach during AAV process development, says Gu. Consequently, the availability of robust analytics that enable the definition and quantification of process CQAs is crucial, Ungari notes.

Multiple serotypes

Today, most cell lines are developed to be effective at producing numerous different AAV serotypes and a broad array of genes of interest. Achieving this goal is an active area of research, according to Tomkowicz. He points to both upstream and downstream processes designed to enable the production and purification, respectively, of multiple AAV serotypes.

Such broad applicability, however, does not preclude the need for optimization. Indeed, Youssef notes that several factors can affect the versatility of a proprietary cell line. He highlights the fact that capsid proteins from different serotypes bind partially to different cellular proteins, which can result in sequestration of host cell factors required
for efficient AAV productivity or in increased levels of impurities; different serotypes may use different RNA splice sites; and serotype-specific leaky promotors may express toxic gene products, requiring specific gene-silencing approaches. “For these reasons, it is crucial to always test the cell line with the specific AAV vector intended for production to ensure high titers and to be prepared with mitigation strategies when specific products turn out to be challenging,” he concludes.

Optimization may also be needed for specific combinations of serotypes and genes of interest, according to Goncalves. The main reasons include the interplay between characteristics of the host cell, AAV serotype-specific capsid interactions, and the molecular properties of the gene of interest. It is also possible that different serotypes have specific characteristics that can interfere with the productivity. These issues may potentially be overcome through optimization of the cell-culture conditions, media formulation, supplements used, or transfection protocols, she says.

One approach to the issue of multiple serotypes posed by Ungari is to adopt “plug-and-play” platforms using the same cell line for all serotypes. “This approach facilitates a rapid and streamlined production process, allowing for efficient interchangeability between different AAV serotypes without the need for extensive modifications to the cell line or production process,” she states. Further optimization specific to a serotype or GOI may still be needed, however, through adjustment of various process parameters using a design-of-experiment approach.

Cost and access

The concept of proprietary technology often raises questions about whether its proprietary nature results in higher cost and/or limited access. In the context of cell-line development, there is no agreement on this matter.

Some believe the development of proprietary cell lines has positive implications for access. “A variety of commercially available cell lines offered through non-exclusive licensing provides AAV manufacturers the option to use validated, high-performing cell lines to support expedited pathways to pre-clinical and clinical studies,” Gu believes.

Ungari concurs. She notes that as a contract development and manufacturing organization (CDMO), AGC Biologics actively develops proprietary cell lines that seamlessly integrate with its production platforms, are meticulously designed to align with GMP standards, and are inclusively provided within the batch pricing structure. Simultaneously, the company uses cell lines developed by its clients, often with some fine-tuning, and offers clients the option of elevating their R&D cell banks to GMP status. “This flexibility underscores our commitment to providing comprehensive solutions that cater to the diverse needs and preferences of our partners within the biopharmaceutical industry,” she comments.

Others see the potential for restriction of access to AAV production. “These cell lines are typically owned by private companies, which may not make them available to other researchers or manufacturers,” notes Youssef. Intellectual property protection grants exclusive rights to the owners for commercial use and makes it impossible for others to use those cell lines without permission and/or payment. Smaller companies, meanwhile, may find cell-line development a cost-prohibitive activity, limiting their ability to pursue innovation that could provide significant productivity and quality benefits. Furthermore, Youssef comments that it becomes challenging for researchers to share and collaborate on their work if only a few companies have access to the best cell lines.

“The cell line is a critical component of the manufacturing process, so at the very least, any proprietary cell line is likely to have restrictions on use and distribution,” Cody states. Some proprietary cell lines also come with licensing fees or milestone payments. “For a gene therapy product developer, the key is to decide if the cost of those fees is offset by potential savings on manufacturing cost due to higher productivity and/or some kind of benefit to product quality,” he observes.

The potential result, Tomkowicz summarizes, is the possible reduction in openness and collaboration in the field of AAV manufacturing, with the focus more on individual proprietary advancements rather than communal development.

On the other hand, Tomkowicz acknowledges that proprietary cell lines can “massively decrease” the cost of AAV production. In particular, providing access to packaging/producer cell line technologies is a perfect symbiosis for a CDMO like SK pharmteco that offers AAV and lentiviral vector manufacturing services. “While the use of these cell lines might involve licensing fees or other costs associated with using patented technology, combining the use of proprietary technology along with scalable GMP-processes as a platform technology is immensely cost-effective for the investigator as well as the manufacturer,” he explains.

At the same time, if certain higher performing cell lines are proprietary, those without access to them could be at a competitive disadvantage, potentially stifling innovation and limiting the diversity of approaches in AAV vector production, according to Tomkowicz.

Therefore, Tomkowicz concludes that “it is a delicate balancing act between providing for the patient, yet generating revenue in the form of IP [intellectual property] to support the business and further drive innovation.”

Continuous development

Despite these issues, ongoing development and innovation in cell-line development for AAV manufacturing is expected to continue, with advances having overall positive impacts across the sector. “There will be increasing interest in proprietary cell line development in the future, as there is always pressure to reduce the cost of production, and increasing productivity is one way to make manufacturing more efficient,” Cody observes.

“The development of proprietary cell lines is an ongoing and continuous process that never stops,” Ungari adds. She also notes that the competitive nature of the biopharmaceutical industry motivates companies to stay at the forefront of innovation, and continuous development allows companies to maintain a competitive edge, ensuring that their proprietary cell lines offer superior performance and efficiency compared to existing alternatives. “Moreover,” she says, “advancements in reagents, technologies and AAV knowledge offer new tools for optimizing cell lines, enhancing AAV production efficiency, stability, and consistency.”

There are, adds Goncalves, several positive aspects in the development of proprietary cell lines. In addition to the investment in new technologies and innovation, she points to an increase in the development of specialized applications that expand the use of AAV therapies. “Developing cell lines tailored for specific therapeutic targets helps to enhance the accuracy and effectiveness of gene therapies, ultimately accelerating the path to clinical applications,” she explains. “New proprietary cell lines,” Goncalves continues, “also help in reducing production costs and contributing to strategic collaborations that foster exchange of knowledge and provide access to proprietary technologies.”

Ascend, in fact, considers proprietary cell-line development the most promising approach to increase cell-specific and volumetric productivity of AAV manufacturing platforms in a disruptive manner, according to Youssef. “By adopting a balanced approach to proprietary cell-line development, we can help ensure that AAV gene therapy remains a promising and accessible treatment option for patients with various diseases. Furthermore, we believe that cell-line development will be an enabler for bringing AAV gene therapy to patients suffering from more prevalent diseases at affordable costs of goods,” he comments.

About the author

Cynthia A. Challener, PhD, is a contributing editor to BioPharm International®.

Article details

BioPharm International®
Vol. 37, No. 3
March 2024
Pages: 10-15


When referring to this article, please cite it as Challener, C. Proprietary Cell-Line Development for High-Titer AAV Manufacturing. BioPharm International 2024 37 (3).