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The growth in demand of viral-vector-based gene therapies drives continuous efforts to improve viral vector manufacturing.
Improving viral vector manufacturing for gene therapy applications is an important step in developing effective end product. In recent years, the biopharmaceutical industry has seen a flurry of activity in which service providers, such as contract development and manufacturing organizations, have invested in expansions of viral vector manufacturing capacity or in enhancements to viral vector development technologies. But why has it quickly become so important to improve manufacturing and development processes for viral vectors?
For one, there have been significant advancements made over the past 10 years in the use of viral vectors within the gene therapy and gene-modified cell therapy fields, explains Arvind Srivastava, PhD, technical fellow, Avantor. For instance, eight therapies using viral vectors have been recently approved by FDA in that timeframe. “Although the early approvals of viral vector-based therapies were for the treatment of rare diseases, more recent approvals have been therapies for cancers of the blood. This shift requires the expansion of global viral vector manufacturing to meet the demands of a larger patient population. For this reason, improvements in the efficiency of viral vector production, as well as refinement of complex production processes, are required to support this growing market,” Srivastava points out.
The ultimate goal of the cell and gene therapy (CGT) field is to deliver new therapies to patients who have few or no other options, continues James Cody, associate director technical sales and evaluations, Charles River Laboratories. Manufacturing is one of the key bottlenecks for these products, both in terms of the time required and the cost, which can be prohibitive in some cases. By improving manufacturing processes, however, the industry can increase overall yields. “This could potentially allow for lower costs and/or smaller batches with more rapid turnaround times. By saving time and cost, we can deliver more therapies to more patients and can do so more rapidly,” Cody emphasizes.
Though relatively new, the CGT space has expanded rapidly since the first human gene therapy trial—using gammaretrovirus—occurred in 1990 for treating severe combined immunodeficiency (SCID). With advancements in molecular biology and biotechnologies enhancing their safety, the massive potential of CGTs to improve patients’ lives has been realized by the biopharma industry, adds Bill Vincent, founder and executive chairman, Genezen. Reflecting this, Vincent points out that the global CGT market was valued at $4.99 billion in 2021 and is expected to grow to $36.92 billion by 2027, at a compound annual growth rate (CAGR) of 39.62% (1).
“The surge in both growth and demand in the CGT space has necessitated rapid improvements in viral vector manufacturing processes—a decade or so ago, the industry was still in its infancy. But, as critical tools in gene delivery, viral vectors need to be produced while balancing quality and speed in order for essential therapies to be delivered to patients quickly,” Vincent says, reinforcing Cody’s sentiment.
In addition to manufacturing processes, regulations surrounding viral vector manufacturing for CGTs have been under pressure to evolve just as rapidly. “But this naturally takes time as the therapeutic area is relatively new. With expertise and experience in the area, it’s fallen to viral vector manufacturers to ensure processes are optimized for safety, speed, and quality. Applying their understanding and knowledge to continually improve processes will mean that they are prepared for future changes to regulatory requirements,” Vincent explains.
Megan Hardy, senior staff scientist, research and development, viral vector services, pharma services, Thermo Fisher Scientific, adds that continuous improvement of viral vector manufacturing processes is necessary as the industry continues to grow and apply lessons learned from the vectors currently being produced. Increased yields and scalability allow for more patients to be treated from a single batch, thereby reaching more of these (often rare-disease) patients sooner.
Hardy’s colleague, Samira Shore, director, research and development, viral vector services, pharma services, Thermo Fisher Scientific, states that improvement of viral vector manufacturing process means not only improving the chance at curing life-threatening diseases, but doing so while minimizing the risk of experiencing off-target responses. “This improvement is focused on maximizing yield of target therapeutic per batch, while maintaining high quality standards and target key characteristics of the product,” Shore says.
Limitations to viral vector manufacturing have included the finite amount of manufacturing space available, says Cody. Many manufacturers have responded to this issue by expanding capacity. Another issue is that many of the key raw materials used for vector manufacturing have lead times measured in months. “These long lead times were an issue prior to the COVID-19 pandemic and have worsened due to increased (and reprioritized) demand as many resources have been diverted to vaccine manufacturing,” Cody says.
“The most difficult challenges in viral vector manufacturing originate in the fine-tuning of complex processes to optimize yield and recovery of vectors,” says Srivastava. In the upstream, processes must be optimized to produce high yields of vectors because it is these processes that define the quality of the end product, he points out. Vector titer can be impacted by plasmid design, cell culture conditions, and key reagents, such as transfection reagents and cell lysis solutions, in upstream processing.
In the downstream process, Srivastava continues, a key challenge lies due to the formation of empty and “false full” vectors in the upstream process that require separation from the full capsids used in the therapeutic end-product. “Separation can be improved using anion exchange chromatography, although the exact operating conditions require significant optimization,” he suggests.
Shore explains that, during the infancy stages of gene therapy, the majority of innovators were developing processes capable of supporting the product yields associated with the target diseases and dosing regimens. Since then, however, the use of viral vectors has expanded for various indications and, lately, viral vectors have been applied to vaccine candidates. The latter brings viral vectors into the arena of high dose, high patient population indications, according to Shore.
This move into a high dose, high patient application (i.e., vaccines) requires, in return, manufacturing processes that can be executed at large scale and yield significant viral vector product to support the therapy treatment needs. “Process scale-up and/or evaluations of suspension-based systems has been a key driver toward overcoming those challenges. Some of the processes, for example, transient transfection or adherent-based systems, are still facing challenges with scale-up and the ability to maintain process control and consistency while expanding to larger scale,” Shore says.
Shore also points out that yield differences are observed when switching from one platform to another. Even if the process is scalable and producing consistently, some innovators are still faced with the regulatory filing challenges of introducing significant process changes post-early phase clinical trials. In addition, there certainly has been much more focus on improving and enhancing analytical methods available to support product characterization, especially with a focus on more quantitative methods, according to Shore.
“There have been several challenges in the past that have hindered the use of viral vectors, the impact of which we are still seeing today,” Vincent says. Notably, safety concerns surrounding the use of gammaretrovirus in CGTs were a considerable obstacle to the expansion of this therapy space. Vincent explains that gammaretroviruses are an attractive CGT tool because of their ability to carry a large cassette, enable the production of stable producer cell lines, and offer high rates of transduction (2). However, these viruses also have an inherent preference for integration near oncogene promoters, increasing the risk of cancer in patients receiving the CGT.
“With this risk in mind, developers moved away from therapies using gammaretrovirus, opting for alternative, safer vectors such as lentivirus. In time, researchers rediscovered the potential for gammaretrovirus in CGT, focusing on other uses where oncogene promoter-adjacent insertion is less of a concern, including CAR-T [chimeric antigen receptor T cell) therapies—and this is where the industry stands now, at an exciting nexus point where potential is becoming reality,” Vincent remarks.
CAR-T therapies are now the most common technology in genetically modified cell therapies, representing 50% of those in the development pipeline (3), according to Vincent; however, the previous safety concerns have meant that expertise in the development and manufacturing of gammaretroviruses is limited. Thus, those organizations offering these capabilities are set to experience a period of significant demand.
In the past five years, the expansive potential of viral vectors as tools for gene transfer in CGT has stepped further into the spotlight. Vincent points to the fact that, currently, five of the 14 FDA-approved CGTs use gammaretroviral vectors (3). “The biopharma industry’s growing interest in this sector has resulted in the global viral vector market being predicted to grow at a CAGR of 18.5% between 2020 and 2026, from a value of $450.5 million to $1.2 billion, respectively,” (4) he cites.
As increasing numbers of CGTs relying on viral vector technologies enter the development pipeline, process development, analytical development, quality control, good manufacturing practice (GMP) cell banking, and GMP vector manufacturing requirements also continue to expand. “It is no surprise that we are seeing a wave of facility expansions and enhancements across the industry in response to this surge in demand,” Vincent says.
Genezen, for example, recently completed construction in April 2022 at its site in Indianapolis, Ind., of an early phase clinical current good manufacturing practice (CGMP) manufacturing facility. The completed facility is just one part of a planned 75,000-ft2 lentiviral and retroviral process development and CGMP vector production facility, Vincent specifies. In addition, the company has announced a next stage of expansion to support the increased demand for GMP viral vectors (5).
The most prominent and most recent enhancement that the viral vector manufacturing industry has implemented is the development of standardized platform processes, observes Hardy. She explains that these platforms aim to shorten process development timelines by leveraging standardized, scalable processes, which have been proven across multiple scales and serotypes, such as in the case of adeno-associated virus (AAV).
“Customers can expect a more ‘plug-and-play’ approach with little, if any, process optimization prior to CGMP manufacture, thus reducing time to clinic and often cost. This approach is especially attractive to early phase customers,” Hardy says. “The industry has also seen tremendous investment in expanding manufacturing capacity, with several key players adding new or expanding existing facilities to meet capacity demands.”
Shore adds that, in addition to process improvements and capacity expansion, significant progress has been made on product purification technologies as well as increased focused analytical methods and instruments.
Outside of new product introductions, Avantor is investing considerably in capabilities that support its viral vector customers, according to Srivastava. This includes expanding internal CGT expertise within the company’s product R&D teams as well as the company’s commercial teams and leadership. “Additionally, we are investing in our global facilities, having recently announced a new distribution center in Dublin, Ireland, as well as a new manufacturing and distribution center in Singapore to support the ever-growing European and Asia-Pacific viral vector markets,” says Srivastava.
With strong demand forecasted over the next several years for viral vector-based CGT’s, it is not surprising that the biopharma industry is continuously seeking ways to improve viral vector manufacturing. As part of the industry’s continuous efforts at improvement, vendors have brought innovation to available technologies to better support the unique requirements of viral vector manufacture, remarks Hardy. These innovations include the advent of the iCELLis technology for adherent cell culture and chromatography resins specifically designed for viral vector manufacture. “Additionally, next-generation analytics such as ddPCR [droplet digital polymerase chain reaction], AUC [analytical ultracentrifugation], SEC–MALS [size-exclusion chromatography–multi-angle light scattering], and CE–SDS [capillary electrophoresis–sodium dodecyl sulfate] allow for more accurate and precise absolute quantification,” Hardy states.
Srivastava emphasizes the point that improvement of viral vector processes requires the fine-tuning of what has traditionally been mAb manufacturing processes. “While some parts of the process are innovative, others are iterative of other bioprocess workflows. In both cases, improvements in manufacturing are being found through the appropriate design of processes with cost efficiency, process efficiency, quality, and compliance in mind,” Srivastava says.
Furthermore, end-to-end planning also mitigates risk across the whole development and manufacturing workflow, Srivastava adds. Such planning enables the identification of strategies to improve process efficiency and vector yield.
“In my opinion, there may not be one single technology that will be the ‘best’ way to improve manufacturing,” says Cody. “Rather, it might be a synergistic combination of improvements across multiple fronts: improved upstream processes with higher cell densities, higher per-cell vector productivity, and improved chromatography materials and methods with higher recovery, etc.”
Meanwhile, Vincent points out that bioreactors—particularly single-use fixed-bed bioreactors—have revolutionized scalable viral vector production. He explains that lentivirus production predominantly relies on adherent producer cell cultures, which are traditionally grown in 2D systems such as cell-culture chambers. Scaling would then involve using this cell-culture chamber setup in multitudes (i.e., scaling-out). “This not only requires huge amounts of incubator space but also extensive manual procedures, increasing the risk of contamination,” Vincent says.
Fixed-bed bioreactors, in comparison, offer a high surface-area-to-volume ratio for adherent cell growth within a compact space. “Their automation and single-use properties also alleviate the need for delicate and time-consuming manual operations, thereby reducing contamination risk. This innovative technology has effectively solved many of the issues associated with scaling lentivirus manufacturing to commercial levels,” Vincent explains.
The development of single-use technologies, overall, has been particularly enabling for viral vector manufacturing processes. Besides fixed-bed bioreactors, other single-use technologies that have been essential in viral vector manufacturing include bag filling and draining systems. “As viral vectors are inherently unstable from a pharmaceutical perspective, exposure to adsorptive containers like glass vials that are often used in fill/finish processes can result in vector loss. Using single-use bag filling and draining systems reduces this potential loss while offering the advantage of continuous filling and high filling volumes for increased speed,” Vincent adds.
1. Research and Markets, Cell & Gene Therapy Market—Global Outlook & Forecast 2022–2027, Market Report (January 2022).
2. X. Wu, et al., Science 300, 1749–1751 (2003).
3. American Society of Gene + Cell Therapy, Gene, Cell, & RNA Therapy Landscape: Q3 2021 Quarterly Data Report, asgct.org (2021).
4. Research and Markets, Viral Vector Manufacturing - Global Market Trajectory & Analytics, Market Report (February 2022).
5. Genezen, “Genezen Completes CGMP-Lentiviral and Retroviral Vector Clinical Production Facility Buildout,” Press Release, April 26, 2022.
Feliza Mirasol is the science editor for BioPharm International.
Vol. 35, No. 8
When referring to this article, please cite it as F. Mirasol, “Driving Manufacturing Improvements Through Viral Vector Advances,” BioPharm International 35 (8) 18–21 (2022).