Manufacturing of recombinant adeno‐associated viruses using mammalian expression platforms

Abstract

Manufacturing practices for recombinant adeno-associated viruses (AAV) have improved in the last decade through the development of new platforms in conjunction with better production and purification methods. In this review, we discuss the advantages and limitations of the most popular systems and methods employed with mammalian cell platforms. Methods and systems such as transient transfection, packaging and producer cells and adenovirus and herpes simplex virus are described. In terms of best production yields, they are comparable with about 10 -10 vector genomes produced per cell but transient transfection of HEK293 cells is by far the most commonly used. For small-scale productions, AAV can be directly purified from the producing cell lysate by ultracentrifugation on a CsCl or iodixanol-step gradient whereas large-scale purification requires a combination of multiple steps. Micro/macrofiltration (i.e. including tangential flow filtration and/or dead-end filtration) and chromatography based-methods are used for large-scale purification. Purified AAV products must then be quantified and characterized to ensure quality. Recent purification methods and current analytical techniques are reviewed here. Finally, AAV technology is very promising, but manufacturing improvements are still required to meet the needs of affordable, safe and effective AAV vectors essential for licensing of gene therapy clinical protocols.

Evaluation of life cycle defective adenovirus mutants for production of adeno‐associated virus vectors

“…Although our A549‐based stable AAV2 producer cell line allowed for a side‐by‐side comparison of the Ad wt and the Ad ∆pTP/ ∆100K mutants as potential helper viruses for a scalable AAV2 vector manufacturing process, the cell line showed strongly reduced productivity after cryo‐conservation, likely as a result of genomic instability or epigenetic silencing. AAV2 vector yields were also significantly lower than titers generally obtained by helper virus‐free transient AAV2 vector production in 293 T cells . Nevertheless, potency as a helper virus, in particular of Ad ∆pTP instead of Ad wt, was demonstrated in the present study.…”

“…On the one hand, one major method currently employed for large scale AAV vector production is an insect cell line‐based platform using baculovirus infection . On the other hand, the most commonly used mammalian cell‐based method for manufacturing both research grade and clinical grade AAV vectors is based on the chemical transient transfection of human HEK293 cells . Manufacturing platforms based on adenovirus (Ad) as a helper virus consist of certain essential components: an AAV vector transgene vector carrying the expression cassette flanked by AAV inverted terminal repeats (ITRs), AAV functions consisting of regulatory (rep) and structural (cap) genes and adenoviral helper functions [E1A, E1B, E2A DNA‐binding protein (DBP), E4orf6 and VA RNA I and II] .…”

Advancements in the design and scalable production of viral gene transfer vectors

“…However, contamination with replication competent adenovirus complicates downstream processing. To date no one has succeeded in creating a stable producer line, speculated to be due to leaky expression of cytotoxic helper virus proteins (Qiao, Li, Skold, Zhang, & Xiao, 2002;Robert et al, 2017). These viruses express sufficient quantities of helper virus proteins to allow vector production, but are attenuated in their own reproduction (Jenny, Toublanc, Danos, & Merten, 2005).…”

Human Embryonic Kidney 293 Cells: A Vehicle for Biopharmaceutical Manufacturing, Structural Biology, and Electrophysiology

“…Despite their broad application purposes, some disadvantages of these cells are that they easily transform into other phenotypes due to heterogeneous and unstable karyotypes and hence result in different glycosylation patterns [Fliedl et al, 2015;Stepanenko and Dmitrenko, 2015]. Moreover, adherent HEK293 cells often require growth in serum, which not only results in increased production costs but also in difficulties to scale manufacturing up [Robert et al, 2017]. Recently, a new strategy has been developed in HEK293 cells by the human cytomegalovirus intermediate-early enhancer promoter in combination with the IFNα2 signal peptide to enhance heterologous protein expression and secretion [Román et al, 2016].…”

“…The use of HEK293 cells was initially limited due to their high investment costs in production applications [Robert et al, 2017]. Nowadays, HEK293 cells are widely used in several areas and have become a powerful vehicle and a new platform for the production of proteins, vaccines, anticancer agents, and recombinant adenovirus vectors.…”