Abstract
The insect cell-baculovirus expression vector system is an established method for large scale recombinant adeno-associated virus (rAAV) production, largely due to its scalability and high volumetric productivities. During rAAV production it is critical to monitor process parameters such as Spodoptera frugiperda (Sf9) cell concentration, infection timing, and cell harvest viabilities since they can have a significant influence on rAAV productivity and product quality. Herein we developed the use of dielectric spectroscopy as a process analytical technology (PAT) tool used to continuously monitor the production of rAAV in 2 L stirred tank bioreactors, achieving enhanced control over the production process. This study resulted in improved manufacturing robustness through continuous monitoring of cell culture parameters, eliminating sampling needs, increasing the accuracy of infection timing, and reliably estimating the time of harvest. To increase the accuracy of baculovirus infection timing, the cell growth/permittivity model was coupled to a feedback loop with real-time monitoring. This system was able to predict baculovirus infection timing up to 24 h in advance for greatly improved accuracy of infection and ensuring consistent high rAAV productivities. Furthermore, predictive models were developed based on the dielectric measurements of the culture. These multiple linear regression-based models resulted in correlation coefficients (Q2) of 0.89 for viable cell concentration, 0.97 for viability, and 0.92 for cell diameter. Finally, models were developed to predict rAAV titer providing the capability to distinguish in real time between high and low titer production batches.
Highlights
Recombinant adeno-associated viruses are an ideal candidate gene therapy vector for many diseases, due to their ability to transduce nondividing cells from several tissues maintaining a long-term gene expression. recombinant adeno-associated virus (rAAV) possess low immunogenicity compared to other viral vectors and are resilient to industry manufacturing methodologies, long-time storage, and in vivo administration [1,2].While several biological systems have been adapted for rAAV production, the insect cell-baculovirus expression vector system (IC-BEVS) is very amenable for large scale rAAV production.Insect cells possess scalable and GMP-compatible characteristics, since they can grow in suspension to high cell densities in serum-free conditions [3]
Accurate targeting of cell concentration at the time of baculovirus infection is critical to maintain rAAV titers due to the so called “cell density effect” [12]
The work developed clearly shows dielectric spectroscopy can be used as a process analytical technology (PAT) tool for this system, by allowing accurate infection time determination, and for rAAV production monitoring
Summary
Recombinant adeno-associated viruses (rAAV) are an ideal candidate gene therapy vector for many diseases, due to their ability to transduce nondividing cells from several tissues maintaining a long-term gene expression. rAAV possess low immunogenicity compared to other viral vectors and are resilient to industry manufacturing methodologies, long-time storage, and in vivo administration [1,2].While several biological systems have been adapted for rAAV production, the insect cell-baculovirus expression vector system (IC-BEVS) is very amenable for large scale rAAV production.Insect cells possess scalable and GMP-compatible characteristics, since they can grow in suspension to high cell densities in serum-free conditions [3]. Recombinant adeno-associated viruses (rAAV) are an ideal candidate gene therapy vector for many diseases, due to their ability to transduce nondividing cells from several tissues maintaining a long-term gene expression. While several biological systems have been adapted for rAAV production, the insect cell-baculovirus expression vector system (IC-BEVS) is very amenable for large scale rAAV production. Processes 2020, 8, 1456 on the high recombinant protein production yields achieved and the absence of mammalian-derived products [4]. This combination resulted in several approved products, targeting Influenza (Flublok® ), cancer (Cervarix® and Provenge® ), and at least one rAAV-based gene therapy (GlyberaTM ) [5,6].
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