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.