DOI: 10.5061/dryad.866t1g1v5
title: Data from: Significant differences in capsid properties and potency between AAV vectors produced in Sf9 and HEK293 cells

description: 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
 consideration when choosing an AAV vector production system for clinical
 production.
description: 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 chromatography was performed with an Acclaim PepMap column
 (15 cm long, 300 μm inner diameter) and a Thermo UltiMate 3000 RSLC system
 (ThermoFisher Scientific) coupled to a Q Exactive HF with a NanoFlex
 source (ThermoFisher Scientific). During online analysis, the column
 temperature was maintained at a temperature of 35°C. Peptides were
 separated with a gradient of mobile phase A (MilliQ water with 0.1% formic
 acid) and mobile phase B (acetonitrile with 0.1% formic acid). The
 gradient was run from 4% B to 6% B over 15 min, to 10% B for 25 min (40
 min total), and then to 30% B for 46 min (86 min total).  Samples were
 loaded directly to the column. The column size was 75 cm × 15 µm
 identifier (I.D.) and was packed with C18 media (Acclaim PepMap,
 Thermofisher Scientific). Due to the loading, lead-in, and washing steps,
 the total time for each liquid chromatography tandem-mass spectrometry run
 was ~ 2 hr. Mass spectrometry data was acquired using a
 data-dependent top-20 method on a Q Exactive HF mass spectrometer,
 dynamically choosing the most abundant not-yet-sequenced precursor ions
 from the survey scans (200–2,000 m/z). Sequencing was performed via
 higher-energy collisional dissociation fragmentation with a target value
 of 1e5 ions determined with predictive automatic gain control; isolation
 of precursors was performed with a window of 4 m/z. Survey scans were
 acquired at a resolution of 120,000 at 200 m/z.  The resolution was set
 for higher-energy collisional dissociation (HCD) spectra to 30,000 at 200
 m/z with a maximum ion injection time of 50 ms and a normalized collision
 energy of 30. The S-lens radio frequency (RF) level was set to 50, which
 gave optimal transmission of the m/z region occupied by the peptides from
 our digest. Precursor ions were excluded with single, unassigned, or six
 and higher charge states from fragmentation selection. BioPharma Finder
 1.0 software (ThermoFisher Scientific) was used to analyze all data
 acquired. For peptide mapping, searches were performed using a
 single-entry protein Federal Acquisition Streamlining Act (FASTA) database
 with carbamidomethylation set as a fixed modification while oxidation,
 deamidation, and phosphorylation were set as variable modifications. A
 10-ppm mass accuracy, a high protease specificity, and a confidence level
 of 0.8 for tandem-mass spectrometry spectra were used.
 Mass spectrometric identification of deamidated peptides was
 relatively straightforward; deamidation adds to the mass of intact
 molecule +0.984 Da (the mass difference between -OH and
 -NH<sub>2</sub> groups). The percent deamidation of a
 particular peptide was determined by dividing the mass area of the
 deamidated peptide by the sum of the area of the deamidated and native
 peptides. Considering the number of possible deamidation sites, isobaric
 species that are deamidated at different sites may co-migrate at a single
 peak. Consequently, fragment ions originating from peptides with multiple
 potential deamidation sites can be used to locate or differentiate
 multiple sites of deamidation. In these cases, the relative intensities
 within the observed isotope patterns can be used to specifically determine
 the relative abundance of the different deamidated peptide isomers. This
 method assumes that the fragmentation efficiency for all isomeric species
 was the same and independent of the site of deamidation. This approach
 allows for the definition of specific sites involved in deamidation and
 the potential combinations involved in deamidation.
description: These are .raw files from Thermo Orbitrap.
description: # 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