Viral vectors, extracellular vesicle, and vaccines
With the rise of new modalities such as viral vectors, extracellular vesicles and vaccines, there is a pressing need to continue improving biomanufacturing capabilities of these biologics. Several presentations at ESACT 2022 were devoted to addressing this topic, some of which being reflected in this special issue as a way of recognizing the enormous efforts of the scientific community to date in this area which are likely to further expand in the future as these modalities become an increasingly important part of the biomanufacturing landscape.
Scalable and cost-effective bioprocesses for production of viral vectors for gene and cell therapy are currently in high demand. Thus, developing new, innovative technologies for production of these products is an emerging thrust in biomanufacturing. One of the most used vectors in gene therapy is the recombinant Adeno-Associated Virus, or rAAV, produced in a variety of cell types. As an alternative to traditional transient transfection systems using HEK93 cell lines, Escandellet al . developed a scalable system for rAAV production using a stable HeLa cell line; it proved to be time-efficient and easy to scale-up while capable of generating high rAAV yields and full to empty capsid ratios. Another increasingly important platform for rAAV production is the insect cells-baculovirus expression vector system (IC-BEVS). To understand the variability in insect populations and its impact on rAAV2 titers, Isidro et al . analyzed the Sf9 insect cell transcriptome using single-cell RNA-seq. While transcriptional heterogeneity in Sf9 insect cells prior to infection exists, mainly associated with cell cycle, that is exacerbated upon infection with the differential expression of baculovirus genes and rAAV transgenes. The genes and pathways identified will inform the path forward for cell and process engineering towards improved rAAV2 production. Finally, Yoonet al. reviews the advances made in rAAV bioproduction as well as the challenges ahead in making these therapeutics accessible. Lentivirus (LV) are another important viral vector used in gene and cell therapies for which large scale production remains a major challenge. Aiming to solve this issue, Klimpel et al . developed suspension-adapted stable packaging cell lines in a scalable and serum-free production process, and compared alternative methods to remove doxycycline in order to initiate LV generation. Oncolytic viruses, another emerging modality, shares the same bioprocess limitations as rAAV and LV. Genzel et al . developed an optimized perfusion process for large-scale production of recombinant vesicular stomatitis virus-based fusogenic oncolytic virus (rVSV-OV). Three cell lines (AGE1.CR, BHK-21, and HEK293SF) were evaluated in HCD cultures, with a 15-30-fold increase in volumetric productivities being observed compared to batch process, establishing perfusion as a viable process for large-scale production of rVSV-OV.
In the area of vaccine development, Palomares et al. applied phage display technology to produce epitopes (or mimotopes) that when attached to AAV virus-like particles (VLP) can produce an antibody response to recognize and potentially protect against Zika and Dengue viruses. In another study, Lorenzo et al. described a process to purify HIV-1 Gag VLPs from contaminating host extracellular vesicles using a series of downstream isolation stages including multiple filtration and chromatography steps.
Another emerging modality for delivery of therapeutics are extracellular vesicles produced by mammalian production hosts, and a number of studies explored methods to further improve their capabilities. In one study, Estes et al . identified specific metabolic pathways in HEK293 and CHO-S cell lines capable of improving extracellular vesicle titers using high throughput siRNA screens. Cholesterol biosynthesis was one of those pathways, results showing that the addition of statins to cell culture increased vesicle productivity up to 9-fold. In addition, Belliveauet al . investigated the impact of osmotic and ammonia stress on the microRNA (miRs) within extracellular vesicles (EVs) of CHO cells. While normal culture conditions included higher levels of mir-92a and mir-23a in EVs, stress condition resulted in the enrichment of let-7a, let-7b, and let-7c miRs, which regulate core oncogenes and may alter the balance between cell cycle and apoptosis.