Comparison of highly pure rAAV9 vector stocks produced in suspension by PEI transfection or HSV infection reveals striking quantitative and qualitative differences

Abstract

Recent clinical successes have propelled recombinant adenoassociated virus vectors (rAAV) to the center stage for human gene therapy applications. However, the exploding demand for high titers of highly pure rAAV vectors for clinical applications and market needs remains hindered by challenges met at the manufacturing stage. The production of rAAV by transfection in suspension cells remains one of the most commonly used production platforms. In this study, we describe our optimized protocol to produce rAAV by polyethyleneimine (PEI)mediated transfection in suspension HEK293 cells, along with a side-by-side comparison to our high-performing system using the herpes simplex virus (HSV). Further, we detail a new, robust, and highly efficient downstream purification protocol compatible with both transfection and infection-based harvests that generated rAAV9 stocks of high purity. Our in-depth comparison revealed quantitative, qualitative, and biological differences between PEI-mediated transfection and HSV infection. The HSV production system yielded to higher rAAV vector titers, higher specific yields, and a higher percentage of full capsids than transfection. Furthermore, HSV-produced stocks had a significantly lower concentration of residual host cell proteins and helper DNA impurities, but contained detectable levels of HSV DNA. Importantly, the potency of PEI-produced and HSV-produced rAAV stocks were identical. Analyses of AAV Rep and Cap expression levels and replication showed that HSV-mediated production led to a lower expression of Rep and Cap, but increased levels of AAV genome replication. Our methodology enables high-yield, high purity rAAV production and a biological framework to improve transfection quality and yields by mimicking HSV-induced biological outcomes.

Why Pall’s Allegro™ Stirred-Tank Bioreactor is ideal for viral vector cell culture

“…The data shows rAAV titer increases throughout the culture with maximum titer being observed at harvest. The final rAAV titers were 4.3 x 10 10 gc/mL and 4.8 x 10 10 gc/ mL for the Allegro STR 500 and STR 50 bioreactors respectively (Figure 4), comparable in range to those reported in the literature [5,6].…”

Microfluidic production of nanogels as alternative triple transfection reagents for the manufacture of adeno-associated virus vectors

“…The smaller size of the particles produced at 1 : 1 : 2 may be due to the presence of a lower quantity of the pHelper plasmid, which has a large size of 17.8 kb in Fig. 3 Particle characterisation comparing increasing NP ratios of nanogels (10,15,20,25) produced by microfluidics, to the control PEI-MAX/ pDNA polyplexes produced in standard batch production. All data depict the pDNA weight ratio of 1 : 1 : 3 for pAAV, pDG9 and pHGTI respectively, where each graph demonstrates; (a) particle size (nm), (b) zeta potential (mV) (c) PDI (d) particle concentration ( particles per mL).…”

“…16 Lipofectamine offers high transfection efficiencies, though its use is limited by the considerable costs of reagents for large-scale transfections 16 and in some cases high cytotoxicity. 17 Since the first reporting of complexing PEI with nucleic acids, 18 this approach has been widely used in triple transfection for AAVs, 14,[19][20][21][22] owing to the ease of preparation and reduced cost in comparison to cationic liposomes. Triple transfection with PEI still requires optimisation given its inability to withstand pH changes, high cytotoxicity to producing cell lines 7 and the opportunity for batch-batch variations.…”

Electrophoresis-Mediated Characterization of Full and Empty Adeno-Associated Virus Capsids

“…Note that these are some of the most established techniques currently available for capsid packaging characterization, but other novel approaches have been gaining traction and have shown great promise as alternative analytical platforms such as mass photometry, capillary isoelectric focusing, and Stunner (UV260/280). 36 − 38 …”

“…The robustness of AUC makes it the quantitative golden standard in the field for AAV quantification despite its high volume, concentration, and time requirements. Note that these are some of the most established techniques currently available for capsid packaging characterization, but other novel approaches have been gaining traction and have shown great promise as alternative analytical platforms such as mass photometry, capillary isoelectric focusing, and Stunner (UV260/280). − …”