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Monoclonal antibody and viral vector manufacture share similarities, but vector scale up faces unique challenges.
Scale up in gene therapy manufacturing currently revolves around viral vector scale up. Although some processes are similar to those in monoclonal antibody (mAb) production (e.g., cell culture to produce the viral vectors), the manufacture of viral vectors carries its own challenges, some of which may be addressed based on the history of mAb manufacture.
A look at the similarities between mAb manufacture and viral vector manufacture show that both products rely on a similar synthesis process: a biochemical reaction-based process and the use of cell culture vessels/bioreactors, according to Naveenganesh Muralidharan, senior manager, manufacturing science and technology group, AGC Biologics, and Suparna Sanyal, director of Commercial Development, Cell and Gene Therapy, Lonza.
With the exception of substrates used for their manufacture, viral vectors and mAbs share in common all the other physiological factors for production, such as temperature, pH, dissolved oxygen, and shear (either from mixing or from sparging gas into the bioreactor for oxygenation). In the production of both, the same process monitoring targets must also be addressed, such as minimizing toxic metabolite accumulation during the cell culture process and sensitivity to times when feed must be added as well as the specific rates at which feed must be added, Muralidharan emphasizes.
Other bioprocessing challenges include cell growth strategy and media optimization in addition to pH control, dissolved oxygen control, mixing, and shear stress, Sanyal adds.
However, one unique challenge that viral vector manufacture poses is that—in the case of lentivirus vectors—lentivirus allows for permanent integration of the vector into the host cell DNA, says Bruce Lahn, PhD, chief scientist, VectorBuilder, a US-headquartered biotech company specializing in gene delivery technologies. “This allows for theoretical ‘permanent’ expression of the transgene in the cells,” Lahn notes. Although other viral vectors, such as an adeno-associated virus (AAV) vector, can be more “transient” than lentivirus, meaning that, although AAV-mediated expression of transgenes, for example, has been sustainable for years, the AAV vector does not integrate into the host genome.
“AAV has a significantly lower immune reaction than lentivirus,” says Lahn, adding that “clinical trials with lentivirus were initially unsuccessful due to the significant patient immune response to the lentivirus itself.” Whereas, AAV clinical trials have demonstrated the safety of AAV vectors, which produce a much lower immune response than lentivirus, he explains.
Lahn furthermore notes that the differences between types of viral vectors (e.g., lentivirus vs. AAV) can be both advantageous and disadvantageous, depending on the end goal of the therapy in development. “If you are looking to target only a specific organ or tissue, then AAV would be your choice. For example, a lot of research has been done to target AAV specifically to retinal cells for the treatment of various vision disorders as well as target it to the brain to deliver therapeutic genes for neurological disorders,” he explains.
The decades of experience by the biopharma industry in the scale-up of mAb manufacturing can offer some lessons that have been applied to viral vector manufacturing. “The tools, technologies, know-how, and infrastructure for efficient cell expansion, seed train generation and scaling up production using single-use bioreactors has been well established in current good manufacturing practices (CGMP) processes for mAbs,” says Sanyal. Because of this, best practices such as cell banking, viability, safety and stability assessment of cell lines, cell growth strategies, metabolic analysis, and production parameter optimization can be easily leveraged for viral vector manufacturing, she emphasizes.
“Over the years of experience in mAb manufacturing processes, companies have templated scale-up activities in biologics. However, viral vector manufacturing processes are novel and highly variable from one manufacturer to other,” states Muralidharan. Therefore, what the industry has established thus far with regards to mAb manufacturing is an in-depth product knowledge for defining critical quality attributes and characterizing the critical process parameters of biomanufacturing processes. The industry also holds a detailed understanding of the variability introduced from raw materials and has well-established process operations for mAb process, says Muralidharan. However, this same depth of knowledge has yet to be established for new therapeutic technologies such as in-vitro gene therapies. As such, setting the release specifications for viral vector manufacturing based on the currently limited process and product knowledge of these molecules remains a challenging task, Muralidharan points out.
Other unique challenges associated with viral vector manufacture include the presence of impurities when produced. As Muralidharan explains, viral vectors manufactured from cell-substrate production systems can contain residual amounts of impurities such as host-cell DNA (hcDNA) and plasmid DNA (pDNA). In the final product, the presence of either impurity presents safety risks to treated patients. To address this risk, global regulatory agencies recommend tight, quantifiable limits for DNA impurities for protein therapeutics (10 ng/dose), according to Muralidharan. “However, because of the inherent nature of hcDNA and pDNA encapsidation in viral capsids, FDA permits viral vector manufacturers to minimize the amount and size of DNA impurities based on risk assessment, rather than requiring quantifiable DNA impurity limits,” he states.
Manufacturers, therefore, define their own hcDNA and pDNA specification limits for final products based on risk assessments to ensure patient safety. Such assessments are not necessarily straightforward, however, and require methods and materials that might not be readily available and that are yet to be established, Muralidharan adds.
“The production process for mAbs is largely standardized and cost-efficient with ease of scalability, using suspension bioreactors and stable cell lines,” says Sanyal. The processes used for viral vector manufacture, in comparison, are more diverse and bespoke. “The majority of viral vector production processes utilize transient transfection processes, which are difficult to scale up and require the use of costly plasmid DNAs,” she points out.
Sanyal also brings up the fact that the production of viral vectors for gene therapies is an emerging market and predominantly targets rare genetic disorders. As such, the target patient population is rather small and requires only small amounts of viral vector for the early phase clinical trials, or even in post-commercialization, she says. “For this reason, many therapeutic developers continue to utilize 2D adherent cell culture processes that are faster to implement but not scalable or cost-effective. As the gene therapy market matures, there is an increase in the recent approvals of viral-vector-based products and a concomitant shift towards the adoption of suspension bioreactor-based production processes to meet market demand,” she adds.
Because transferring non-optimized small-scale R&D processes to commercial scale can lead to much uncertainty, according to Muralidharan, and that uncertainty can lead to a high risk of failure to pass through process performance qualification (PPQ) and meeting that validated state consistently during commercial production, it makes sense to have smart steps in place for when scale up moves forward.
“The smart step is to start planning for commercialization as early as the preclinical testing phase. This early planning for commercialization will lead to developing scalable process optimization work during early development. The key for successful commercial process starts there,” says Muralidharan.
Meanwhile, Sanyal points to the transition to a 3D suspension, bioreactor-based process as a key first step in scaling up viral vector manufacturing. The next smart step from there is process optimization for upstream unit operations to maximize viral vector productivity per cell. Following that, subsequent optimization of downstream purification to help maximize yield, purity, and product quality. “The use of perfusion-based processes can also improve production in some cases,” Sanyal says. “Additionally, the use of staggered campaign manufacturing strategies can be utilized to maximize production throughput to meet commercial demand.”
Finally, Lahn discusses the benefit of having a wide variety of AAV serotypes to work with, as AAV serotypes can target specific tissues. As such, “one of the hottest trends in the gene therapy world is the work to create new AAV serotypes that can target specific tissues or cell types. This process can be done by rational design or by creating an extensive library of AAV capsids and screening for capsids that can infect cells/organs more efficiently or infect new cell types,” he explains.
Furthermore, with machine learning and artificial intelligence becoming more prevalent in scientific research, their use has shown great promise in identifying novel AAV capsids. This stands in contrast to the lentiviral system, which cannot be optimized and screened this way with “smart” tools, notes Lahn.
Feliza Mirasol is the science editor for Pharmaceutical Technology.
Volume 47, No.11
When referring to this article, please cite it as Mirasol, F. Gleaning Scale-Up Lessons from mAbs for Viral Vectors. Pharmaceutical Technology 2023, 47 (11), 20–2