Kelly Geosits and Ngan Trinh , associate scientists at SK Pharmteco , share some results from research into adeno-associated viral vectors

In addition , close to 60 % of the 500 + gene and gene-modified cell therapies in clinical trials in 2021 targeted more prevalent diseases . These treatments are generally administered systemically , require large doses and can serve as first-line therapies . 2 , 3

To ensure the long-term success of gene therapies , manufacturers of viral vectors must achieve robust , readily scalable , cost-effective processes . Most direct gene therapies leverage AAV vectors as delivery vehicles , complex products whose functional performance is impacted by numerous biophysical characteristics .

While packaging and producer cell lines provide the most robust and scalable solutions for adeno-associated viral ( AAV ) vector manufacturing , their

Table 1 - Platforms for AAV manufacturing development remains challenging . Coinfection methods using the insectbased baculovirus expression vector system and human-derived herpes simplex virus Type 1 systems are also readily scalable and result in high yields of fully functional vectors , but have extended development timelines , may have quality issues and / or can be challenging to purify .

For these reasons , transient transfection of three plasmids ( rep , cap and helper ) in HEK293 cells facilitated by a transfection reagent is the most widely used method for AAV vector production . It is effective for all AAV serotypes and is a regulatoryvalidated approach .

The fact that transfection occurs over a fairly short time also allows for rapid development of optimum processes and thus shorter times to the clinic and the market . 4 Table 1 lists the platforms available for AAV manufacturing .

Transient transfection involves multiple steps , including cell expansion and growth , formation of the plasmid

DNA-transfection reagent complex and transfer of the complex into the bioreactor at large scale . Viral particles are then generated once the DNA is transfected into the cells and delivered to the nuclei , where it is transcribed and translated to enable expression of the appropriate viral proteins ( Figure 2 ).

Transfection reagents are necessary for transient transfection because naked DNA is not readily transported across cell membranes . Initially , calcium phosphate was used . Next-generation solutions include lipid-based lipofectamine reagents and versions of the linear polymer polyethyleneimine ( PEI ).

More advanced transfection reagents designed specifically for AAV and lentiviral ( LV ) vector production via transient transfection are now available that combine the properties of lipidand polyamine-based compounds for improved performance . These aid not only in the transport of DNA across the cell membrane , but also delivery to the nucleus , and with reduced cytotoxicity . 5

Rep / Cap Plasmid Plasmid Integrated in cell line First rHSV Integrated in cell line

ITR-transgene Plasmid Plasmid BEV Second rHSV Integrated in cell line

Helper genes Plasmid Plasmid BEV rHSVs ( above ) Wt adenovirus

Cell line HEK293 ( adherent ) HEK293 ( suspension ) Sf9 insect cells BHK ( suspension ) HeLa S3 ( suspension )

Production system CellFactory , roller , CellCube Wave reactor ( tens of liters ) 200 L stirred tank reactor 10L wave reactor 250L stirred tank reactor

Scalability - ++ +++ +++ +++

Safety concerns None None None Contaminating helper virus Contaminating wild-type helper virus

Stable producer cell line to produce for each project

32 SPECIALITY CHEMICALS MAGAZINE ESTABLISHED 1981