Contract development and manufacturing organizations (CDMOs) specialized in adeno-associated viral (AAV) vector services must adeptly support a spectrum of projects, from small-scale to large-volume production, while navigating the complexities of upstream process scalability. Viralgen Vector Core, with a complete focus on AAV therapies bolstered by a robust suspension process applicable from research through commercial phases, is uniquely equipped to guide AAV-based gene therapy developers. With experience spanning over 1,000 AAV batches, Viralgen possesses comprehensive expertise in process and analytical development, regulatory navigation, and a digital-first approach that fosters data sharing and collaboration. This positions Viralgen to adeptly address the challenges of manufacturing for both rare and common diseases, ensuring early consideration of manufacturability and quality standards. By seamlessly integrating these components, Viralgen is uniquely positioned to accelerate the journey of AAV-based gene therapies from concept to commercial realization while ensuring patient safety and product quality.
A New Modality Brings Challenges
AAV vectors as delivery vehicles for in vivo gene therapies still represent a new modality compared with more traditional biologic drugs, such as monoclonal antibodies (mAbs) and recombinant proteins. Regulations and the expectations of health authorities for AAV-based therapeutics have not yet been well defined. As such, each new product presents a unique set of challenges because not only must an effective process be established and scaled, but the overall development strategy must be determined on a case-by-case basis.
In addition, most AAV-based gene therapies target rare diseases for which the number of patients is quite limited. Only a few batches are needed, and in some instances a single batch is sufficient for the target population. The strategy for commercialization from development to submission, therefore, must be very well defined, with each step clearly delineated and understood by all stakeholders and clearly accepted by the appropriate regulatory agency. Furthermore, appropriate analytics must be in place that allow for testing using minimal quantities of material to ensure that product quality and patient safety standards are met while conserving sufficient material for patients.
Process variability is another challenge, as AAV vector production involves complex processes with many aspects that are still not well understood or characterized. Each new serotype, whether natural or engineered, and every new project faces its own set of challenges. While there are “platform” process steps that do not require process development from scratch, there is still significant need for product-specific process optimization — first on a small scale (e.g., low-volume reactors or flasks) and further optimized while scaling up to pivotal scale (e.g., 2,000 L). Scaling is not an insignificant challenge, and effective scale-up requires the completion of multiple tasks and studies in single-use bioreactors (SUBs) at various scales.
Different Production Volumes Required for Different Indications
Further complicating AAV vector development is the fact that, as the field has advanced, drug makers have expanded the target indications for direct gene therapies from rare diseases with small patient populations to more widespread diseases with larger patient populations, in addition to some requiring larger and potentially repeated doses. For the former products, only one or two batches may be necessary. For the latter, larger quantities of material are needed, often at higher virus concentrations. Process performance — efficiency and productivity with optimized costs of goods — become very important, as well as yield.
Chemistry, manufacturing, and controls (CMC) strategies are different for different production volumes, and organizations looking to manufacture AAV vectors for both rare and prevalent diseases must be able to meet the differing regulatory requirements for those programs. If only one or two batches will be needed, historical data must be leveraged to set specifications. If multiple batches are produced, current data will be available to establish specifications. For CDMOs, it is also necessary to meet the nuanced needs of each customer, which vary from one to the next.
Regulatory Uncertainties
One of the biggest differences between mAbs and AAV vectors at present is the current degree of regulatory clarity. In the early 2000s, there were guidelines for small molecules, but none yet established for mAbs. Today, there are well-recognized guidelines for biologics, but the equivalent has yet to really crystallize for cell and gene therapies and other advanced therapy medicinal products (ATMPs).
Current guidelines are based on those established for mAbs, for which one can produce many large batches within a shorter time frame, with lower costs compared with AAV. That does not really fit the current situation for AAV production. More guidelines are therefore needed that adapt the existing strategy to the current needs. One example would be guidelines for performing process performance qualification (PPQ) having only a single batch rather than the traditional three required for mAbs.
Achieving effective guidelines will require extensive collaboration between gene therapy developers, AAV manufacturers and regulatory authorities.
Several Factors Influence Variability During Scaling
Scaling requires the performance of both mathematical modeling and physical experiments. Some process parameters are scale-independent and must be maintained as the process scale increases, while others must be adapted to the scale of the bioreactor. Mathematical modeling is important for predicting the optimum values for scale-independent parameters, while physical experiments, typically using a design-of-experiment approach, are necessary to optimize scale-dependent factors.
Another important challenge to scaling AAV manufacturing is the complexity of the transfection process. In the case of mAbs, the antibodies being produced are similar to proteins naturally expressed by cells. With transient transfection to generate AAV vectors, three different plasmids enter the cell to generate the viral capsid containing the gene of interest (GOI). This process called transfection is the critical process step during the AAV production and needs to be optimized for every project and used bioreactor size, since various factors, like the size of the GOI, the time of transfections, and the ratio of the three plasmids, may impact the efficacy (amount of product AAVs) of the AAV production process and the quality of the final product (impacting the number of capsids not containing the GOI or only containing partial genetic sequences).
The Right Analytics are Essential
The fact that a large number of the AAV capsids generated via transient transfection are empty or contain only partial genetic sequences is a significant issue, as it is known that capsid content impacts the efficacy of AAV-based gene therapies. Implementation of effective analytics that allow for comprehensive measurement of the different forms (e.g., full, empty, partial) is therefore essential. It can also be challenging, as there are few commercially available methods for evaluating full versus empty capsids, and some of those that are on the market are not GMP compliant yet (like charge-detection mass spectrometry (CDMS)) or have some GMP hurdles (like analytical ultracentrifugation (AUC)).
Advances in analytical technologies are expected to improve capabilities in this respect. AUC for full/empty capsid analysis has become the gold standard during research and development, and some companies are now implementing this methodology under GMP conditions. Mass photometry is attracting interest as well, as it is easy to use and can be applied in both quality control and GMP settings. Widespread use would reflect a paradigm shift in AAV testing.
Potency testing presents another area of concern, as the mode of action of AAV-based gene therapies is very complex, and the link between analytical results and clinical outcome is difficult to demonstrate. However, it is necessary to ensure product potency and efficacy, which requires the use of reliable and robust potency methods.
Application of Data-Based Prediction for Scaling
The over 1,000 lots of AAV vectors produced at Viralgen have generated considerable historical data that are leveraged by the company’s digital science team, using internal digital tools to make predictions of batch performance through modeling. This information has enabled Viralgen to achieve linear scalabilities from 50 liters to 2,000 liters. Currently, the manufacturing, science, and technology (MSAT) team is qualifying various small-scale upstream processing (USP) and downstream processing (DSP) models to work on process characterization studies that promote successful and smooth PPQ activities and robust and consistent processes during commercial production.
Benefitting from Digitalization
Digitalization is essential for improving the efficiency and productivity of all aspects of CDMO businesses. As a young and dynamic company, Viralgen has a strong Data Science department that is making digitalization a priority across many different business and manufacturing activities, which adds tremendous value to the company.
For instance, technology transfer is implemented using QbDVision software, which makes it possible to speed up tech transfer activities and perform failure mode and effects analysis (FMEA) risk assessment and process characterization according to quality-by-design (QbD) principles. which ultimately facilitates smooth and efficient tech transfers, whether processes are transferred from customers into clinical or commercial manufacturing or within Viralgen from clinical to commercial production. This ensures that processes are thoroughly understood and robust before beginning PPQ) and subsequent commercial production.
Another example is digitalization of laboratory data. Lab notebooks used by the MSAT and Development groups will no longer be physical containing hand-written notes but digitalized with well-established connection to analytical and production equipment. In this manner, it will be possible to easily share digital “real-time” data both internally within Viralgen and with customers provided access to the system.
Collaborative Approach to AAV Development and Manufacturing
One of the strengths of Viralgen is the collaborative approach taken by the company. Interactions between the MSAT and the quality assurance, regulatory, process and analytical development, and production groups helps ensure consistent, high-level performance. As projects progress, representatives from all relevant groups work together, providing the different perspectives of each of those groups along with comprehensive support. This collaborative approach is greatly appreciated by customers, who benefit from the close internal interactions, which enable efficient and effective support throughout the whole life cycle of the process from first feasibility runs up to production in the pivotal commercial production site.
Collaboration is not only emphasized internally but also with external groups. That includes the pursuit of excellent relationships and open communication with various regulatory authorities, including the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA).
Expanding Capabilities with a New MSAT Facility
As a bridge linking the development and commercial production groups, MSAT serves as an important facilitator for projects, not just as they transition from development to production but from the outset, ensuring that manufacturability issues are taken into consideration as early as possible. The group not only uses its own USP, DSP, and analytical labs to perform a wide range of tasks. These activities include process characterization, addressing scale-up challenges, and conducting investigations into deviations. Furthermore, the facility has a dedicated team that specializes in ensuring smooth and efficient tech transfers from development or early clinical production sites — whether internal or external — to pivotal commercial sites. This team is adept at responding swiftly to support development needs while also providing a high level of support for commercialization activities and ongoing commercial production.
In recognition of the crucial role that MSAT plays at Viralgen, a new facility will be opening soon that will include state-of-the-art MSAT USP and DSP process labs which will mirror the commercial production setup using both upstream and downstream small-scale models (from 250-mL Ambr® bioreactors to 50-L SUB) to support process characterization, process improvement activities, and preparations for PPQ.
Notably, the production and analytical equipment used in the small-scale lab will be from the same vendors that supply larger-scale equipment to Viralgen. This will assure linear scalability between 50-L SUBs and the reactors used for clinical and commercial production (up to 2,000 L). The same analytical instruments and validated platform analytical methods are used by the MSAT, Development, and GMP-QC groups to ensure that data can be easily and reliably compared across different testing stages (e.g., stability, process characterization, process contamination, and release).
Focused on a Flexible Suspension Platform
Viralgen focuses fully on AAV vectors and has developed extensive expertise in their production. The company supports suspension-based AAV vector manufacturing and employs the Pro10TM cell line developed by AskBio across R&D and manufacturing. Using the same cell line and media across scales has allowed Viralgen to gain extensive process experience, evident in our ability to perform robust production across various scales, which has created an excellent database with historical process data that can be used further for process optimization, specification settings, and other CMC-related purposes.
Since 2018, the company has produced more than 1,000 batches using nearly all available serotypes and numerous GOIs at different scales. We have learned to deal with these differences, have developed and validated both platform and serotype-specific analytical methods, and have used all gathered data to predict process behaviors. Viralgen’s dynamic and motivated teams use that information to implement effective processes at the company’s clinical and commercial manufacturing facilities.
This provides our customers with comprehensive support from development to drug substance and drug product production, thru to fill/finish, as well as QC and regulatory support, all of which is tied together through our MSAT group, serving as a bridge between all relevant departments and assuring the successful commercialization of customer products.
