Improving AAV Manufacturing Process with Design of Experiment and Fit for Purpose Transfection Reagent
Introduction
The gene therapy field has exploded in recent years. At the end of 2022, at least 24 gene and gene-modified cell therapies had received marketing authorization worldwide. Five of these were approved in the U.S. that year, the most ever in a single year by the U.S. Food and Drug Administration (FDA). Eight more gene and gene-modified cell therapies could receive FDA regulatory decisions in 2023.
Nearly 1,100 clinical trials investigating gene and gene-modified cell therapies were underway in mid-2022. Approximately one-third of those trials involved direct gene therapies, nearly 75% of which rely on recombinant adeno-associated viral (rAAV) vectors for delivery of the genetic material. Of the 1100 trials, the delivery vector was public information for 45%, with lentivirus predominating (48%), followed by rAAV (26%). Despite these clinical advances, therapeutic vector manufacturing remains a challenge.
AAV vectors are preferred for the direct delivery of gene therapies because they are not pathogenic and elicit only minimal immunogenic reactions in animals and humans. Targeting a wide range of tissues is also possible with access to numerous wild-type and engineered rAAV serotypes, and rAAV can deliver genetic material to many different dividing and non-dividing cells. AAV-based gene therapies are consequently in development for a variety of indications, including ophthalmic, neurological, metabolic, hematological, musculoskeletal, and cardiovascular diseases.
The cost to develop, manufacture, and deliver rAAV-based gene therapies remains a significant issue, however. Transient transfection processes are often inefficient and unoptimized due to the rush to be first to market with these potentially curative treatments. Typical lab-scale adherent cell-culture processes that involve numerous manual interventions are lengthy, not readily scalable, and generally impractical to implement in a GMP environment. High raw material costs and low yields for viral-vector production are two additional problems that must be resolved.
These factors contribute to a cost of goods for gene therapy manufacturing ranging between $500,000 and $1 million —which must be reduced if rAAV-based gene therapies for the treatment of more prevalent diseases and larger patient populations are to be commercially viable and accessible. As importantly, due to the current process limitations, scaling out and scaling up to the needed volumes is insufficient to meet expected growth in demand at an acceptable cost-per-dose.
Scaling out is accompanied by a linear increase in costs with the use of multiple production trains based on the same inefficient processes and is not a long-term solution. Scaling up to larger bioreactors does reduce operating costs, but again gains are limited by the inherent inefficiency of most rAAV manufacturing processes. Process optimization and intensification must be realized in combination with the development of more readily scalable processes.
Due to the complexity of transient transfection processes and the fact that numerous and varied factors can directly impact process performance, one of the most effective methods for establishing optimum solutions is through a design-of-experiment (DoE) approach. Process intensification, meanwhile, can be achieved using fitfor-purpose plasmids and transfection reagents.
The Benefits of a DoE Approach
Development of an optimal process cannot be achieved without having a deep understanding of the process, including the various factors that can influence yield, quality, and other performance attributes. A DoE approach provides a systematic and efficient mechanism for gaining deep insight into the critical process parameters (CPPs) that directly influence critical quality attributes (CQAs) (Fig 1). In addition, a well-designed DoE study reveals crucial information about the interactions of those CPPs and potential non-linear effects, knowledge that cannot be accessed using one-factor-at-a-time (OFAT) studies.
Fig 1. While the OFAT approach can resolve one parameter at a time, by keeping all the other parameters constant, a DoE approach enables simultaneous assessment of multiple parameters in a timely manner.
For transient transfection processes, using a DoE approach leads to optimum processes that maximize cell and system performance by identifying the sweet spot where requirements for excellent cell growth and viability are achieved in combination with high transfection efficiency and generation of a high level of functional vectors.
In addition, because the design space is well understood and normal operating ranges have been clearly determined for these optimized processes, they are also robust with predictable performance, making them readily scalable.
For production of rAAV vectors via triple transient transfection, there are numerous potential CPPs that must be considered when developing a DoE study strategy. Process performance can be impacted by the medium, cell line and cell density, as well as the temperature, pH and dissolved oxygen content during cell culture. Plasmid design, total plasmid quantity and plasmid ratios, as well as the choice of transfection reagent and quantity employed may influence transfection efficiency.
Specific outcomes that are often evaluated during DoE studies of AAV manufacturing processes include the functional and viral genome titers, full/empty capsid ratio, the presence of encapsulated plasmid DNA and host-cell DNA impurities, other product-specific CQAs, and cost-per-dose.
The goal is to vary the values for numerous inputs simultaneously and evaluate a range of outputs to find the best set of process conditions that meet the desired outcomes with respect to titer, quality, and cost.
To successfully achieve this goal and develop a robust, optimized AAV manufacturing process, five important actions must be taken:
Clearly defining the goals for a DoE study and selecting appropriate factors for evaluation based on previous experience with transient transfection processes
Choosing the right design for the intended purpose that fits with the defined goals and takes into consideration the available capabilities. Often choices must be made given a specific timeline and resources
Preparing the experimental plan must be done in a manner that minimizes variability
Understanding and developing the right analytical methods that will enable consistent and reliable evaluation of the target outputs
Iterating to confirm results is highly recommended, including looking at both larger and closer ranges to ensure coverage of the full design space
While DoE studies do involve upfront expenditures and can seemingly extend development timelines, the benefits of process optimization often far exceed these initial investments. Vector yields can often be increased many folds within just a few months, providing purer products that require simpler downstream processing, leading to dramatically reduced costs and often shorter run times.
Successful Customer Scaleup
Sartorius Polyplus® has been working with numerous CDMOs and gene therapy developers to upscale AAV transient transfection processes using proprietary transfection reagents. These partners include Andelyn Biosciences, Catalent, Center for Breakthrough Medicines, Pall (now Cytiva), FinVector, Exothera and LogicBio, among others.
One collaboration between Sartorius Polyplus, Exothera and LogicBio achieved the scale up of an AAV process to the 2000-L scale with no loss of titer. The suspension-based processes were performed in Pall Corporation (now Cytiva) Allegro™ STR single-use bioreactors (SUBs) and afforded an AAV product with a titer of approximately 8 x 1011 VG/mL at all scales (shake flasks and 1-L, 40-L, 200-L and 2000-L SUBs).
This successful scale up of a complex transient transfection process could not have been achieved without a deep understanding of the process. That knowledge was gained by using the DoE approach to process development. Similar results have been achieved by other customers committed to leveraging DoE studies to develop optimized, robust, high-performing processes.
Process Optimization with Engineered Raw Materials
The benefits of performing effective DoE studies can be magnified by using fit-for-purpose reagents designed and engineered specifically for AAV manufacturing. Plasmid engineering can provide DNA of the highest quality that contains only necessary genetic material. Appropriately crafted transfection reagents can boost transfection efficiency while increasing complex stability and reducing complexation volumes.
FectoVIR®-AAV transfection reagent is an example of a fit-for-purpose raw material. This compound was specifically designed for use in suspension-based transient transfection processes for scalable production of AAV vectors. In addition to providing a two-fold increase in AAV titers compared to Sartorius Polyplus’ polyethyleneimine (PEIpro®) reagent and a 10-fold increase in titer compared to other PEI-based reagents on the market, FectoVIR®-AAV transfection reagent exhibits this improved performance at complexation volumes of 5% (and in some cases as little as 1%) of the culture volume, compared to the typical 10%. Complexes of FectoVIR®-AAV transfection reagent and plasmid DNA are also much more stable, allowing longer transfer times if necessary for more fragile complexes. Consistent performance is observed at small to large scale, and FectoVIR®-AAV transfection reagent is available in research and GMP grades.
Building a Toolbox for Process Intensification and Scaling of AAV Production
Gene therapy developers and CDMOs involved in AAV manufacturing are faced with the challenge of developing more efficient and productive processes that provide consistent, high performance from the lab to commercial production, including at ever-larger scales. Realizing this goal is possible by using a DoE approach and purposefully designed and engineered reagents.
Sartorius Polyplus® is your comprehensive partner for optimizing and intensifying AAV processes. Our expertise extends beyond offering the cutting-edge FectoVIR®-AAV transfection reagent, we provide plasmid engineering and Design of Experiments (DoE) services. We are committed to collaborating with you to develop customized DoE plans that integrate our transfection reagents and plasmids. Our team will expertly implement these plans, conduct detailed data analysis, and present the findings, ensuring the success of your project.
References
Alliance for Regenerative Medicine, “Regenerative Medicine: The Pipeline Momentum Builds,” September 2022.
Citeline (Previously Pharma Intelligence) and American Society of Gene + Cell Therapy, “Gene, Cell, & RNA Therapy Landscape Report: Q4 2022 Quarterly Data Report,” https://asgct.org/publications/landscape-report
Erin Harris, “Breaking Down Pricing Of Cell & Gene Therapies,” Cell & Gene, June 18, 2019. https://www.cellandgene.com/doc/breaking-down-pricing-of-cell-gene-therapies-0001
BioInsights - FectoVIR®-AAV, the new tool to enhance recombinant AAV production for gene therapy.
