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The manufacture of gene therapy vectors is shifting to more modern technologies.
With an increasing number of gene therapy candidates expected to move through the clinical trial pipeline and toward commercialization, the need for innovative process development to bring these potential new products to commercial scale is a focus in the biopharmaceutical industry. Among the bioprocessing steps seeing forward movement is the cell culture for producing adenovirus-associated virus (AAV) vectors for gene therapy, which has largely depended on less efficient methods of cell culture but is moving to more modern methods.
Currently, many methods used for AAV production still involve adherent cell culture, meaning that cells are grown on a solid surface, such as flasks or multi-layered cell culture dishes or roller bottles. These traditional systems are generally difficult to scale-up and are associated with a high propensity of mishandling risks, which are the result of increased processing time due to the manipulation of large numbers of culture vessels, notes Alengo Nyamay’Antu, PhD, scientific communication specialist at Polyplus-transfection, a France-based biotechnology company specializing in gene and cell therapy, biologics manufacturing, and life-science research. Some direct consequences of following these traditional cell culture methods during manufacturing scale-up are compromised reproducibility and space limitation problems, Nyamay’Antu says.
To achieve larger production volumes for AAV vectors, scale out rather than scale up has been typically employed, says Jerry Keybl, Head of Cell & Gene Therapy Manufacturing, MilliporeSigma. Scaling out uses specialized equipment that increases the surface area of the flask so that a greater number of cells can attach.
“Typically HEK293 cells are grown in undefined media, such as DMEM [Dulbecco’s modified eagle medium] that contain fetal bovine serum. Unfortunately, the manual handling of such culture formats increases contamination risk and is extremely costly and labor intensive,” Keybl states.
Going from small-scale to commercial-scale production presents several challenges. For example, scale up requires a significantly large number of vessels, which results in an increase in the number of incubators and space needed to house them, says Susan D’Costa, senior director, Technical Program Design, Commercial Operations, Viral Vector Services, Thermo Fisher Scientific. In addition, scale-up requires an increase in the resources needed to manipulate the cell culture vessels. The risk to asepsis is also higher because of the number of manipulations needed, D’Costa asserts.
Scaling to commercial-level production runs into technical difficulties as well, such as adjusting the process for large-scale production without impacting production yield and reproducibility, Nyamay’Antu points out
An alternative to scaling out of traditional cell culture methods, such as flasks, is the use of bioreactors. Bioreactors are becoming more frequently used, even for small batches of vector product, observes Jorge Blanco, director of operations at VIVEbiotech, a Spain-based biotechnology company. Bioreactors simplify the production process, increase reproducibility, and reduce costs associated with footprint and labor, Blanco notes.
Because traditional cell culture methods operate within two dimensional (2D) systems that can be only a few cm2 or multi-layered stacked cell factories, they present poor scalability, Blanco confirms. For reaching the required commercial yields, therefore, multiples of 2D cell factories are required. “This makes these systems far less operational,” Blanco states. “A very high number of multi-layered cell factories are required to reach the yields that are easily harvested from single 3D adherent bioreactors. Additionally, these 2D cell factories require high manual handling, thus lowering reproducibility significantly.”
The fact that many cells are adherent, meaning they have to be attached to a substrate in order to grow, significantly limits scaling up, Ulrich Kettling, PhD, chief business officer, CEVEC Pharmaceuticals, further explains. “When it is not possible to use larger dishes to provide larger surfaces, the only option is to scale out.”
Keybl attributes scaling challenges to the fact that the flasks are limited by surface area, and the only way to increase production is to add more flasks and incubators to the process. “Up to hundreds of flasks and incubators may be utilized at one time to meet the demands for a commercial product. Subsequently, the increase in resources to handle and process the flasks are proportionately increased as well,” he states.
The move toward modern cell culture methods and techniques would mitigate the challenges and limits inherent totraditional methods when scaling for commercial production.
Two major trends of alternative culture systems that address scalability and reproducibility are fixed-bed cell culture systems that increase adherent cell seeding density and volumetric productivity and suspension cell culture systems that provide the needed flexibility to adapt a production scale to meet large-scale production demands. “We are clearly seeing that alternative systems are taking over, and already at small scale, as viral vector manufacturers are increasingly anticipating what their process should already look like at small scale to facilitate transition to large-scale manufacturing,” Nyamay’Antu says.
“When it comes to AAV manufacturing, there is a clear preference for suspension cell-based systems, directly inspired by the success of this approach for commercial-scale manufacturing of recombinant proteins and antibodies produced in mammalian cells,” observes Nyamay’Antu. For instance, suspension cell systems improve batch-to-batch reproducibility during scale up by eliminating varying cell culture parameters (e.g., serum, cell seeding density, etc.) and by simplifying downstream AAV purification.
“In addition to improved reproducibility, with suspension cell systems it is possible to further increase AAV production yields with the right set of tools: cell lines that can be grown at higher density, optimized set of plasmids, the transfection reagent, and the synthetic medium used to support AAV production,” Nyamay’Antu adds.
“Some processes are beginning to use a fixed-bed bioreactor, but these processes still require the use of serum, introducing adventitious agents and consistency challenges,” Keybl chimes in.
VIVEbiotech, meanwhile, uses the adherent production system for cell culturing a different type of vector: lentiviral vectors. The company uses fixed-bed bioreactors, however. “Today, scaling up is feasible given the availability of intermediate scales in the market, which allow manufacture from early stages to commercial. It is achievable to get up to 1200 liters of harvest from fixed-bed bioreactors,” Blanco says. “Additionally, different providers for fixed-bed bioreactors are already on the market, de-risking the chance of running out of them or of increasing cost of consumables.”
The smallest bioreactor sizes are adequate for process development, Blanco also notes. These small bioreactors can easily perform fine-tuning runs without investing high sums of resources, he adds.
“Regarding downstream processing (DSP), fixed-bed-based manufacturing turns into simpler DSP processes, as once the viral vectors are bulk harvested, DSP processing requires fewer steps than for suspension systems,” Blanco explains.
Each technology has strengths and weaknesses, Blanco further enumerates. It is well known, for instance, that viral proteins are cytotoxic for producing cells regardless of the manufacturing technology used. However, there are some additional facts that increase the stress to which the cells are exposed, he highlights. “In stirred-tank reactors, producer cells are exposed to mechanical forces that may result in a higher cell lysis during the growth within the bioreactor. Any stressing factors may directly impact functionality, and thus performance of the resulting viral product,” Blanco states.
“Adherent viral vector production systems do also present some aspects that need to be addressed. The growth of producer cells in absence of animal-origin components, such as fetal bovine serum, is more challenging than in suspension systems. VIVEbiotech’s R&D department is able to produce vectors in the absence of serum in 2D productions, and tech transfer to reactors is currently ongoing with the aim of offering serum-free 3D productions in the near future,” continues Blanco.
“Last but not least, the industry is moving towards continuous bioprocessing. The ‘go continuous’ tendency is especially challenging in viral vector manufacturing because, as mentioned, viral vectors affect the viability of producer cells due to the toxicity of virus-producing proteins. This is why cell cultures, and thus, harvesting time, cannot be lengthened as wished,” Blanco adds.
Suspension bioreactors are the format of choice as commercial production platforms for biotherapeutics. “Suspension bioreactors in volumes up to tens of thousands of liters and stable producer cell lines have been used in industry for decades now, for manufacturing of recombinant proteins and monoclonal antibodies (mAbs),” Kettling states. “And this way of manufacturing is now becoming available for gene therapy vectors as well, including innovations in stable producer cell lines combined with high cell density-processes such as perfusion.”
Gene therapy commercial production can benefit by taking a lesson from the mAb production world. “Scaling up cell expansion by growing cells in suspension culture in controlled bioreactors is certainly the wave of the future and where the industry is moving to,” says Keybl. “Suspension methods are scalable, less labor intensive than scaling out adherent production, and can utilize chemically defined media, which is a huge benefit to downstream purification/patient risk.At MilliporeSigma, we have a number of production platforms in development to accelerate adoption. These processes are in development across the industry and I believe will power gene therapy manufacturing in the future.”
“Adherent cell culture can be scaled up into micro- and macro-carrier-based reactors,” adds D’Costa. “However, the better alternative is to adapt adherent cells into suspension cells grown in large scale bioreactors in serum-free, chemically defined media. Transient transfection of large-scale reactors is still limited to 200–500-L scale, so currently the process needs to use multiple 200–500-L reactors that are harvested at the same time. While traditional purification methods utilizing cesium chloride/iodixanol density gradients for enrichment of AAV full particles are currently used in commercial production, more scalable and process-validatable methods include purification through chromatography media (capture and polishing),” D’Costa explains.
Regardless of the method for cell culturing viral vectors, however, there are two viral-vector production modes: transient transfection and stable producer cell lines, Blanco points out. Although each production mode has its own pros and cons, there is a general trend leaning toward stable producer cell lines. The cost associated with plasmids used for transient transfection is very high, for example, which increases the final price of the product considerably, Blanco explains. Stable producer cell lines, however, allow dispensing with these DNA molecules, which thus makes scaling-up much smoother and translates to a decrease in expenses and an increase in reproducibility. “The generation of stable producer cell lines is a long and challenging process that requires the involvement of significant material and personnel resources,” he says.
Modernizing the cell culture methods and techniques for vector production, whether AAV or lentiviral, also poses its own challenges. The biggest challenges for a company like VIVEbiotech, which has already spent more than 30 months fine-tuning the manufacturing process, include improving the performance of the final product (i.e., the ratio of empty to active infectious particles) so that the lentivectors are more functional and increasing recovery, Blanco states.
In terms of performance, however, there are increasingly more transfection enhancers bringing promising results, Blanco adds. “As for the recovery after DSP processing, it is usually around 30% when using adherent culture systems. This means there is still considerable room for improvement, so VIVEbiotech´s development professionals are working tirelessly on this, already achieving recoveries of 40%.”
Transient transfection has particularly been a challenging area when scaling to commercial production, Kettling affirms. Issues with reproducibility and robustness of the transfection steps, as well as the significant costs for GMP-grade plasmids, require solutions, Kettling observes.
The adaptation of the cell line to the suspension process is another major challenge on the path toward modernization, says Keybl. “Typically, adherent cells need to be carefully adapted into different types of cell culture media (preferably serum- and animal-component-free) and selected for good growth and viral vector productivity. This can mean several years of R&D development time in order to choose the right cell line and medium combination that will give the optimal results,” he states. “Commercially available cell lines are coming to market and can reduce this burden for innovators.”
Meanwhile, bridging the data obtained with traditional methods, including non-clinical toxicology and early clinical data, to conform to newer commercial-grade processes presents a significant challenge for those that want to change over to modern methods, D’Costa points out. “It is also important to consider the additional development time it takes to change the upstream production and downstream purification process and refine the assays, which is needed to build out tools for cell adaptation,” she says.
“When aiming to switch from traditional adherent to suspension cell systems, there are several parameters that need to be considered,” includes Nyamay’Antu. “I would not consider them as challenges that need to be overcome, but more as part of protocol optimization. As for all new set-ups, it requires delving into the literature, relying on the technical support of suppliers, and timescales. Some of these questions include whether to adapt the cells to suspension or to buy a commercially available suspension cell line; which synthetic medium should cells be grown in, and whether it is the same during the production phase as well as which transfection reagent will reach highest production yield in these suspension cells. These are all valid questions when moving on to suspension cell systems, and during the upscale process additional questions rise when choosing the set-up for large scale culture (bioreactor),” Nyamay’Antu emphasizes.
To ease the transition from older cell culture methods to more modern means requires a number of breakthrough innovations. Recent innovations in materials, such as media/supplements that support high density cell culture, transfection reagents that support high productivity, and single-use bioreactors that are scalable, have made the shift from older methods to modern methods easier, says D’Costa.
Particularly important is the development of producer cell lines to meet the high industrial demands on the production processes: stable producer cell lines offer reliable and consistent quality combined with sufficient yields in standardized processes and equipment, Kettling points out. Several labs and innovative biotech companies are working on providing solutions, including Cevec, which recently launched a new producer cell-line technology (ELEVECTA) in April 2020 (1). The ELEVECTA technology enables large-scale manufacturing of AAV vectors using fully stable producer cell lines and can be implemented in bioprocess development and large-scale GMP manufacturing facilities that use standard suspension bioreactor equipment.
“Thankfully, suppliers have been working hard to support the transition of AAV manufacturing in suspension cell culture systems. We can see that companies that develop synthetic cell culture medium, supplements, and also plasmid DNA now offer tailored tools specifically for AAV production,” Nyamay’Antu (Polyplus) chimes in. Polyplus-transfection, for example, has been aware that viral manufacturers needed a transfection reagent specifically developed for large-scale transfection to improve scalability, productivity, and flexibility of AAV manufacturing in suspension cells. The company developed a chemical-based and animal-free transfection reagent (FectoVIR-AAV) that addresses current limits of transfection for AAV manufacturing in suspension cells.
“These limits include scalability, flexibility and, last but not least, productivity as transfection, whether at small or large scale, is dependent on the efficiency of the delivery molecule,” Nyamay’Antu says. “FectoVIR-AAV improves AAV production yield in suspension cells up to 10-fold in functional titer yields compared to [other reagents]. The increased production yield is reproducible at different scales for the production of several AAV serotypes as confirmed by viral manufacturers who took part in the beta-testing.”
On the supplements and reagents front, VIVEbiotech is working to introduce some new items to its production process, such as a transfection agent. “Given that animal-origin reagents need to be avoided in medicines to be applied in humans, the standard benzonase has been removed from the production process and replaced by an animal-origin-free endonuclease, with really good results. [We] are also working hard trying to move to serum-free media for producer-cell-culturing,” Blanco states.
1. CEVEC, “Cevec Announces the Launch of the ELEVECTA Platform—the Stable Producer Cell Line Technology for AAV Gene Therapy Vectors,” Press Release, April 28, 2020.
Feliza Mirasol is the science editor for Pharmaceutical Technology.
Vol. 44, No. 10
When referring to this article, please cite it as F. Mirasol, “Modernizing Bioprocessing for Gene Therapy Viral Vectors,” Pharmaceutical Technology 44 (10) 28–33 (2020).