Despite the undeniable ability of viral vectors to deliver genetic payloads, and despite the incredible promise of this approach to treat a vast variety of diseases—including metabolic, cardiovascular, muscular, hematologic, ophthalmologic, and infectious diseases as well as cancer—hurdles remain, especially in manufacturing.
Due to its complexity, viral vector production is still evolving. Experts concur that overall yields of functional capsids containing the desired genetic payload is the biggest obstacle. Most often, transient transfection is used, essentially creating a single-run “cell factory,” that is, a cell line for each manufacturing run. A stable and optimized production cell line, like those used for other biologics, would provide a more robust process, and it would be scalable if cells were grown in suspension.
Progress is being made, but new technologies and approaches to standardize processes take time to develop and implement, especially if the processes involve GMP-grade reagents. To accelerate progress, developers are implementing new tools. Six of these developers are discussed in this article. Each has insights to offer on ways to streamline production.
Scalable host cell lines
Viral vectors are complex—significantly more complex than traditional recombinant protein therapeutics. Besides consisting of both proteins and nucleic acids, viral vectors present daunting production challenges. For example, production processes for viral vectors typically incorporate lipid- or polymer-based transfection reagents and lysis buffers, production components that are not needed in stable, recombinant protein–expressing production systems.
According to Jonathan Zmuda, PhD, director of cell biology, Thermo Fisher Scientific, viral vector production involves challenges both upstream and downstream. Typical challenges include scalability issues, difficulties maintaining productivity, and overall lack of robustness.
Significant progress has been made in recent years to address scalability. Yet the commonly used HEK293 cells remain less well characterized for large-scale production than are their protein expression CHO cell counterparts.
In contrast, Sf9 (Spodoptera frugiperda) insect cells are well validated for cost-effective adeno-associated virus (AAV) production at 1000-L scale or greater; however, insect production systems possess workflow steps with which many laboratories are unfamiliar. Thus, transient transfection of HEK293 cells remains the most efficient way to generate AAV.
“The producer cell line forms the foundation of any viral vector expression system,” says Zmuda. “Great care must be taken to ensure that the appropriate host cell line is chosen at the earliest stages of development.” Shortcomings of the HEK293T adherent platform include scalability and the requirement for animal serum, whereas the presence of the oncogenic large T antigen impacts both adherent and suspension HEK293T platforms.
To address the cell line challenges posed by AAV production, Thermo Fisher developed CTS Viral Production Cells (VPCs) for the production of scalable, high-titer lentivirus in suspension HEK293 cells. More recently, Viral Production Cells 2.0 (VPCs 2.0) were developed specifically for AAV production. VPCs 2.0 is a clonal HEK293 cell line selected for its ability to support production of high-titer and high-quality AAV in stirred-tank bioreactors via transient transfection in the Gibco AAV-MAX Helper-Free AAV Production System, which is currently available as a prototype.
Contract development and manufacturing organizations (CDMOs) like Thermo Fisher’s Pharma Services business are a viable option for gene therapy developers that need viral vectors. CDMOs have experience with industrial-scale gene therapy workflows and can provide reliable and efficient production processes.
The yield challenge
The largest hurdle in viral vector production is yield. All cell lines have inhibitory sequences that influence vector production. And much remains unknown about the viral capsids themselves. For example, viral capsid proteins may ungergo different post-translational modifications in different manufacturing processes. Some critical structural features may need to be preserved to ensure in vivo potency.
“If yield could be doubled, it would be possible to reduce costs by a factor of two,” says Thomas VanCott, PhD, global head of product development at Catalent’s cell and gene therapy business unit. Catalent was one of the first facilities to produce the AAV vector and the first FDA-licensed CDMO for gene therapy products.
VanCott suggests that developers may be unable to make enough AAV capsids per cell or enough functional capsids. Another difficulty is that the transfection process is complicated and requires optimization. Although to some degree the process is transferrable, each plasmid system is a little different. Plasmid DNA, the core ingredient, is manufactured by just a few companies.
“They do it well but cannot meet demand,” VanCott asserts. “To stay on timeline and to have uniformity throughout the process from research lots through GMP grade, we have invested in bringing that capability in-house. Also, we recently announced the acquisition of Belgium-based Delphi Genetics.”
In downstream processing, the functional capsids must be purified away from the empty ones. Ultracentrifugation works well but is labor intensive and slow. Although different chromatographic techniques perform better, a more efficient approach using continuous flow would be of value in addition to improved analytical methods to measure the percentage of empty versus full capsids.
“Viral vectors must be intact and carry the gene of interest,” VanCott emphasizes. “Our platform minimizes the parameters that need to be modulated to lock in a process quicker. The key gets down to securing the supplies with the visibility of lead time.”
Materials, such as single-use bioreactor bags, media, and other reagents for manufacturing, may be hard to secure. The manufacture of COVID-19 vaccines uses similar supplies and has priority in the supply chain.
Proprietary cell lines
For gene therapy, the delivery workhorses are AAV, adenovirus, and lentivirus. If they are to be produced efficiently, manufacturers need a suitable cell line. “Ideally, a stable cell line should be used to minimize batch-to-batch variation,” says Jeffrey Hung, PhD, chief commercial officer, Vigene Biosciences. “But cell lines are very tricky to produce and maintain because of some inherent toxicity of the viral genes when they integrate into the cell genome.”
Vigene has developed mammalian and insect cell lines for virus manufacturing in either suspension or adherent culture. The company notes that the characteristics of the selected cell line (origin/derivation, doubling time, and permissiveness for viral infection and replication) determine the efficiency of viral productivity, whereas the growth conditions determine the requisite downstream processing methods and release tests for the final drug product.
Because insect cells are smaller, more of them can be packed into a set volume. And they offer higher yield for AAV culture. Vigene’s insect cell line technology uses the baculovirus expression system to produce AAV vectors in Sf9 cells under serum-free conditions. However, this is not a plug-and-play technology. Once the AAV is packaged, the baculovirus must be purified away.
For other cells, such as HeLa cells, removal of any residual cellular DNA must be demonstrated. Each cell line presents challenges.
“There are many opportunities for improvements in cell lines,” notes Hung. For example, cell lines could offer better packaging efficiency and greater stability. They could address the potential harms associated with residual DNA. They could also facilitate gains in manufacturability and scalability.
Vigene Biosciences supports AAV, adenovirus, lentivirus, and retrovirus production. In addition, the company offers services to support “everything from basic research to launching a commercial product.”
“A viral vector is just a means to an end,” Hung adds. “Our customers can be anywhere in the development cycle—at the conceptual stage or at mid-scale or clinical-grade production. Sometimes customers have produced certain preclinical-grade products. These customers will find that it is advantageous for them to keep the same cell platform. This option is available for production continuity.”
Purification and analysis
Process optimization is complex. Upstream scientists must cope with multiple culture and transfection parameters, whereas downstream scientists must cope with the absence of standard platforms that work for all vectors, serotypes, and expression systems. Scientists face some very specific challenges, such as the separation of empty and full AAV capsids, as well as the need to adapt the downstream bioprocess for each serotype.
Complex analytical methods and requirements take time to establish. Processes need to be sufficiently productive in terms of overall efficiency, yield, purity, and characterization to ensure robustness to increase the chance of regulatory success as well as derisk GMP production campaigns.
“We have invested heavily in our high-throughput platforms, such as the Ambr platform, for cell culture, in combination with our design of experiments software,” says Amélie Boulais Raveneau, head of market entry strategy, Viral-Based Therapeutics, Sartorius Stedim Biotech. “This enables manufacturers to take a systematic approach toward optimizing cell culture and transfection parameters.”
The recent acquisition of BIA Separations adds that company’s “monoliths,” monolithic chromatography columns, to the Sartorius portfolio. The monoliths have unique chemistries that allow for high-resolution AAV purification. Sartorius also gains a development laboratory that develops AAV processes.
“We have a platform that can be optimized to rapidly purify all serotypes of AAV and enable efficient empty/full capsid separation,” Boulais Raveneau notes. “The platform does not rely on expensive AAV affinity assays. Instead, it utilizes a combination of orthogonal chemistry to achieve the level of purity and yield required.”
The BIA acquisition also helps Sartorius offer scientists an expanded toolbox of reliable technologies to measure critical quality parameters and performance attributes. For example, BIA’s CIMac analytical chromatography columns are efficient tools for quantification and for getting the full picture of the sample through the fingerprinting method. Earlier, Sartorius acquired Danaher Life Sciences’ FortéBio business, which brought Sartorius the Octet platform, which allows rapid at-line virus titer quantification to serve as an alternative to the time-consuming ELISA assay.
Processes need to be suitable for GMP production. They also need to be standardized. The fulfillment of these needs is being facilitated by the transition to suspension processes and by research into AAV-agnostic purification trains.
New manufacturing platforms
The continued use of adherent cultures, legacy cell lines, and unoptimized unit operations means that developers or their CDMOs need to devote additional time to resolving yield and efficiency issues. Even experienced developers tend to rely on CDMOs to resolve these issues and bring processes to commercial scale. CDMOs, however, have capacity limitations, so there is a bottleneck of available slots for production. Furthermore, not every CDMO is a good match for every developer in terms of process capabilities.
“MilliporeSigma is taking a comprehensive approach to meeting the need for more efficient viral vector production,” says Angela Myers, the company’s head of gene editing and novel modalities. “To address technical challenges, we created the VirusExpress Lentiviral Production Platform, which combines a high-performance packaging cell line with a chemically defined medium and optimized suspension process to enhance the scalability of upstream operations.
“Along with our work to develop templated processes for lentiviral and AAV production, and a forthcoming VirusExpress platform for AAV, we strive to create the new platform standard for viral vector manufacturing. To address industry logistical challenges, we recently invested in a $121 million expansion of our viral vector manufacturing CDMO facilities in Carlsbad, CA. Implementing our product technologies will allow us to manufacture vectors better and faster.”
Coming online at the end of 2021, the new facility will accommodate 1,000-L single-use bioreactors to add both scale and production slots to service the growing need for high-quality viral vectors. The increased recognition of the need to transition to suspension production demonstrates that more of the industry is not only recognizing the challenges of traditional vector production, but also thinking ahead about meeting commercial scale and regulatory needs.
Furthermore, technology developers are creating fit-for-purpose tools to address unit operation challenges unique to viral vectors. There is a strong desire to proactively adopt the lessons learned from the commercialization of other biologics over the past three decades.
Quality evidence of clonality
A single-cell cloning stage is necessary in viral vector production. “Clonality relates to a stage in the manufacturing process where regulatory bodies require quality evidence that the process has been brought down to a single cell to minimize the heterogeneity within the cell bank to limit process variability,” says Duncan Borthwick, PhD, global marketing manager, Solentim. “Our technology is used to assure clonality in the production of cell lines and also to increase the efficiency of cell growth.”
The Solentim technology, which consists of the company’s VIPS (Verified In-Situ Plate Seeding) and Cell Metric instruments, dispenses single cells and captures images to provide quality evidence of clonality. According to the company, the technology is the gold standard for capturing evidence of clonality for regulatory submission. Users include GlaxoSmithKline, Thermo Fisher, and BrammerBio (acquired by Thermo Fisher in 2019).
VIPS dispenses cells at very low pressure in nanoliter droplets into plate wells. Each cell is imaged in the bottom of its dry well as evidence of seeding. After the well is automatically filled with media, the Cell Metric whole-well cell imager takes over and images at day 0 for a double lock of assurance. Daily imaging continues to determine if the division rate logically matches to that of a single cell, providing additional evidence.
Applying the Solentim technology is an attractive alternative to using manual limiting dilution techniques and statistics. With the Solentim technology, it is possible to accumulate the data needed to make a solid case to regulators. “Getting the clonality stage right is massively important for process engineering,” says Borthwick. “Insufficient data would have a massive impact on timelines and regulatory progression.”
Solentim’s InstiGRO growth supplements assist in more rapid cell growth, helping individual cells survive the lonely process and positively impacting the workflow. For instance, GlaxoSmithKline reported an 11-fold increase in clonal outgrowth. InstiTHAW aids the freezing and thawing process.