Significant differences in capsid properties and potency between AAV vectors produced in Sf9 and HEK293 cells

For successful vector-based gene therapy manufacturing, the selected adeno-associated virus (AAV) vector production system must produce vector at sufficient scale. However, concerns have arisen regarding the quality of vector produced using different systems. In this study, we compared AAV serotypes 1, 8, and 9 produced by two different systems (Sf9/baculovirus and HEK293/transfection) and purified by two separate processes. We evaluated capsid properties including protein composition, post-translational modification, particle content profiles, and in vitro and in vivo vector potency. Vectors produced in the Sf9/baculovirus system displayed reduced incorporation of viral protein 1 and 2 into the capsid, increased capsid protein deamidation, increased empty and partially packaged particles in vector preparations, and an overall reduced potency. The differences observed were largely independent of the harvest method and purification process. These findings illustrate the need for careful ..., Studies were performed as previously described. Briefly, ammonium bicarbonate, DTT, and iodoacetamide (IAM) were purchased from Sigma Aldrich, and acetonitrile, formic acid, trifluoroacetic acid (TFA), 8 M guanidine hydrochloride (GndHCl), and trypsin from ThermoFisher Scientific. Stock solutions of 1 M DTT and 1.0 M IAM were prepared. Capsid proteins were denatured and reduced at 90°C for 10 min in the presence of 10 mM DTT and 2 M GndHCl. Samples were allowed to cool to room temperature and then alkylated with 30 mM IAM at room temperature for 30 min in the dark. The alkylation reaction was quenched with the addition of 1 mL of DTT. 20 mM ammonium bicarbonate (pH 7.5–8) was added to the denatured protein solution at a volume that diluted the final GndHCl concentration to 200 mM. Trypsin solution was added for a 1:20 trypsin-to-protein ratio and incubated at 37°C overnight. After digestion, TFA was added to a final concentration of 0.5% to quench the digestion reaction.
Online chromato..., These are .raw files from Thermo Orbitrap., # Title of Dataset

Differences between Sf9- and HEK293-derived AAV vectors

Tryptic digest of AAV samples.

## Description of the data and file structure

Tryptic digests of AAV vector samples from serotypes AAV1, AAV8, AAV9 from HEK293 production (PD077, PD078, PD079)and SF9 (DG17021, DG17022, DG17023)

## Sharing/Access information

Data was derived from the following sources:

* QExactive

## Code/Software

Data was analyzed using BioPhrama FInder