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The choice of expression system requires wise consideration of product complexities.
Despite the advent of advanced technologies to meet the needs of novel biopharmaceuticals in development, the manufacturing processes for these molecules remains complex and challenging. New issues that are emerging in the upstream processing stage give rise to further considerations in terms of scalability, cost, and process robustness. A closer look at up-and-coming categories of biomolecules in the development pipeline can give further insight into how the industry can best address new and emerging hurdles.
The field of biopharmaceutical development has exploded in the past decade with a variety of modalities, particularly following the regulatory approval success of novel therapeutics such as cell therapies, gene therapies, and bispecific antibodies. Currently, the biopharma industry is focused on several “hot” biomolecules and therapeutic modalities, saysRyann Russell, head of Upstream Process Development at Center for Breakthrough Medicines.
Among the categories of biomolecules garnering attention are chimeric antigen receptor T cell (CAR-T) therapies, RNA-based therapeutics, gene therapies, viral vectors, and oncolytic viruses, Russell enumerates. Furthermore, gene-editing has also become a hot area of focus as gene-editing techniques, such as clustered regularly interspaced short palindromic repeats (CRISPR), have led to new horizons of molecular development.
CAR-T therapies are groundbreaking, Russell emphasizes, in that they have revolutionized cancer treatment. The use of genetically modified T cells with antigen-targeted receptors have given these therapies a means to precisely identify and attack malignant cells, he notes. Gene therapies, meanwhile, fundamentally involve the delivery of genetic material to replace, supplement, or modify defective genes with the aim to treat genetic disorders or certain types of cancers. Within the gene therapy umbrella, multiple approaches are being explored; among them are gene replacement, gene addition, gene silencing, and gene editing, Russell adds.
Russell further explains that RNA modalities are fundamentally about modulating gene expression. “With flexibility in the gene pathways being targeted, there is potential to treat a variety of ailments, including cancer, infectious diseases, and genetic disorders,” he states.
Meanwhile, advances in viral vector technology have resulted in new avenues that the biopharma industry can pursue. For instance, viral vectors derived from modified viruses are used as delivery vehicles to transfer therapeutic genes into target cells. In the case of gene therapy, Russell notes, a viral vector introduces a therapeutic gene of interest that can act to mitigate disease. For vaccine applications, the genetic payload can mimic a pathogen and be delivered in a format that mirrors natural infection, thus leading to a robust immune response, Russell explains. Viral vectors can also be used as part of CAR-T design, making them a versatile component in various new biotherapeutics.
“One exciting aspect of viral vector development involves capsid engineering to improve tissue targeting,” Russell says. “Other hot areas involve optimization of ITRs [inverted terminal repeats] to get around limitations in gene size or to improve promotor function. Vectorology, as a whole, is a very hot topic to improve the capability of gene therapies or to mitigate downsides.”
Oncolytic virus-based therapeutics, meanwhile, involve the use of engineered viruses to specifically target and destroy cancer cells. “Established bioprocessing technologies used for live viral vaccines can be readily leveraged for this modality. Opportunities exist for the design of such viruses to ensure specificity of their cancer targets and mitigate side effects,” says Russell.
New upstream challenges
Currently, cell and gene therapies attract the bulk of attention from biopharma developers, and new manufacturing and technical challenges in the upstream have emerged.
Russell notes areasin the upstream production strategy where these stability and sensitivity challenges pop up, including bioprocess scale up, contamination control, automation and integration, and supply management. For instance, cell and gene therapies require the production of large amounts of material, which can be challenging to scale-up. Manufacturing-scale methodologies that are used must also consider the labile nature of these types of products, he also says.
The complexity of cell and gene therapies make them inherently more prone to contamination. Thus, to minimize or eliminate contamination, rigorous control and monitoring of the upstream bioprocessing stage is crucial. “Avoiding these risks also requires innovative approaches that fit within the constraints of our process needs,” Russell states.
Automation and integration of upstream bioprocessing systems are also crucial for improving efficiency, reproducibility, and scalability in the manufacturing of cell and gene therapies. Automated platforms can perform repetitive tasks with high precision, for example. These automated platforms therefore reduce manual errors and improve process efficiency. In addition, integration of different unit operations through automated systems ensures seamless workflow and data traceability, according to Russell.
Meanwhile, management of the supply chain holds its own challenges. Cell and gene therapies often involve specialized raw materials, such as viral vectors, growth factors, and cell culture media, Russell observes. Thus, ensuring a robust and reliable supply chain for these materials is essential to maintain consistent manufacturing processes and product quality.
Optimizing the upstream process for cell and gene therapies, as well as other new biologic modalities, will rely on the ability to be flexible.In fact,developing optimized and flexible upstream bioprocessing strategies is vital to achieve high yields, reduce costs, and adapt to changing manufacturing requirements, Russell emphasizes. “This includes optimization of cell culture conditions, media formulation, and process parameters. What might seem like subtle differences from one product to the next can have a large impact to the performance of a platform process. As such, we require a flexible approach to each product,” he explains.
Quality control and analytics are other areas of which to be mindful. Robust quality control measures and analytical testing are critical to ensure product safety, identity, purity, and potency. Russell points out that implementing advanced analytical techniques and comprehensive analytics throughout the upstream bioprocessing workflow is essential for process monitoring and product characterization.
Finally, cell and gene therapies are subject to strict regulatory guidelines to ensure patient safety and product efficacy. Thus, compliance with regulatory requirements, such as current good manufacturing practices, is necessary throughout the upstream bioprocessing stage to ensure regulatory approval and successful commercialization. “We need to keep this end goal in mind before work begins and incorporate that early in development,” says Russell.
The biopharma industry’s growing painsfrom ironing out traditional monoclonal antibody (mAb) bioprocessing have imparted certain lessons that can provide insight into solving today’s challenges with cell and gene therapies.
Russell notes that the lessons learned from decades of optimizing mAb manufacture has provided a better understanding of the fundamentals of the unit operations of bioprocessing. This understanding includes approaches to optimizing upstream and downstream operations, improving cell culture media, and developing better methods for cell and gene therapy product characterization.
What’s more, the use of process analytical technology (PAT) and advanced analytics has also been instrumental in monitoring and controlling the bioprocessing of cell and gene therapies and have enabled real-time feedback, Russell adds. With this feedback, bioprocess operators can thus make adjustments to optimize process performance and product quality.
“Lessons learned from mAb manufacturing have highlighted the importance of process robustness, scalability, and quality control. These principles have been applied to the development and optimization of upstream bioprocessing for cell and gene therapies. By leveraging this knowledge, researchers and manufacturers have been able to overcome some of the challenges associated with scaling up cell and gene therapy production,” Russell states.
Furthermore, improvements in cell culture media formulations have played a crucial role in optimizing cell growth and productivity in cell and gene therapy manufacturing. Enhanced understanding of nutrient requirements, growth factors, and culture conditions, for example, has led to the development of specialized media that support the expansion and differentiation of therapeutic cells.
For upstream processing, the choice of expression system for gene therapy production, specifically, is a complex decision. Such a decision depends on the specific product and its use, the desired yield, and timeline, explains Russell.
For antibodies, there is a choice between mammalian and bacterial cells.For instance, mammalian cell lines remain the most common choice for gene therapies, as these cells are able to properly fold and modify the proteins and other molecules expressed. However, bacterial cell lines have been explored as well, and may be a better choice in some cases. “For example, bacterial cells can be used to produce large quantities of proteins quickly and at a lower cost compared to mammalian cells. Bacterial expression systems, such as Escherichia coli (E. coli), have a well-established track record in the production of recombinant proteins for various applications,” Russell further explains.
Russell also advises that when considering the choice of expression system, several factors need to be taken into account including the following:
Protein complexity and post-translational modifications
As is commonly understood, mammalian cells are capable of performing complex post-translational modifications, such as glycosylation. These modifications are crucial for the proper folding, stability, and functionality of certain proteins, Russell states.
“If the therapeutic product requires specific post-translational modifications that are not efficiently performed by bacterial cells, a mammalian expression system may be preferred. Whichever system is chosen, codon optimization may be required to better match the host for more efficient protein translation,” he remarks.
Russell observes that mammalian expression systems typically have lower yields compared to bacterial systems. If high protein yields are essential, bacterial expression systems, such as E. coli, can produce large quantities of proteins more quickly and at a lower cost. Russell notes the importance of considering potential challenges in folding and solubility when using bacterial systems, however, which may require additional optimization steps.
At the end of the day, the gene of interest (GOI) is the therapy, not the proteins, however. As such, implications for efficient packaging of the GOI into the proteinaceous vector must be considered.
Meanwhile, bacterial expression systems, such as E. coli, are generally considered safe for the production of non-toxic proteins, Russell makes the distinction. However, the high amount of endotoxin poses a safety risk as well as processing challenges. “For example, concentration factors required to generate high-dose viral vector therapies can also concentrate even small amounts of endotoxin to unacceptable levels. Efforts to remove endotoxin come at a step-yield loss that erodes the value of the platform. As such, mammalian cell lines or baculovirus expression systems may be more suitable choices due to their ability to perform complex protein modifications and reduce the risk of endotoxin contamination,” he adds.
It is also important to note that regulatory authorities often have specific guidelines and requirements for the choice of expression systems in gene therapy manufacturing. Thus, it is crucial to consider the regulatory landscape as well as ensure compliance when selecting an expression system. As Russell points out, incumbent technologies have already been shown to have a favorable safety profile and, therefore, those systems appear to a lower risk path compared to novel solutions.
The future of upstream processing may further push boundaries. “It is worth noting that the choice of expression system is not limited to mammalian cells or bacteria. Other expression systems, such as yeast, insect cells, or even cell-free synthesis may also be considered based on the specific requirements of the therapeutic product,” Russell states.
In the end, the decision on the choice of expression system for gene therapies should be made on a case-by-case basis, and factors such as protein complexity, post-translational modifications, yield requirements, time-to-market, safety considerations, and regulatory guidelines should all be considered, Russell says. Furthermore, it should be noted that collaboration between process development scientists, molecular biologists, and regulatory experts is often necessary to make an informed decision that optimally balances these considerations, Russell concludes.
Feliza Mirasol is the science editor for BioPharm International.