Analysis of AAV-Extracted DNA by Charge Detection Mass Spectrometry Reveals Genome Truncations.

Adeno-associated virus (AAV) is a widely used gene therapy vector. The intact packaged genome is a critical quality attribute and necessary for an effective therapeutic. In this work, charge detection mass spectrometry (CDMS) was used to measure the molecular weight (MW) distribution for the genome of interest (GOI) extracted from recombinant AAV (rAAV) vectors. The measured MWs were compared to sequence masses for a range of rAAV vectors with different GOIs, serotypes, and production methods (Sf9 and HEK293 cell lines). In most cases, the measured MWs were slightly larger than the sequence masses, a result attributed to counterions. However, in a few cases, the measured MWs were significantly smaller than the sequence masses. In these cases, genome truncation is the only reasonable explanation for the discrepancy. These results suggest that direct analysis of the extracted GOI by CDMS provides a rapid and powerful tool to evaluate genome integrity in gene therapy products.

Analysis of thermally driven structural changes, genome release, disassembly, and aggregation of recombinant AAV by CDMS.

Charge detection mass spectrometry (CDMS) was used to analyze recombinant adeno-associated virus serotype 8 (rAAV8) vectors after incubation at elevated temperatures. rAAV8 vectors with a range of genomes of interest (GOIs) from 2.22 to 4.84 kb were investigated. For the shorter GOIs, GOI release occurred at surprisingly low temperatures (15 min at 45°C for cytomegalovirus [CMV]-GFP). The released DNA and intermediates with the GOI extruded from the capsid were detected. The temperature required to release the short GOIs is well below the 65°C incubation temperature required to disassemble the empty rAAV8 capsid. The temperature for GOI release increased with its GOI length. With the longer GOIs, the GOI stabilized the capsid so that it remained intact under conditions that would disassemble the empty particle. After incubation at 65°C, the main species in the CDMS mass distributions for the longer GOIs was the vector with the GOI. However, for GOIs longer than the wild-type genome (∼4.7 kb), the stability diminished, and genome release occurred at a lower temperature. Heterogeneous DNA fragments from the host cells or plasmids is released at a lower temperature than the longer GOIs, suggesting that the GOIs have a feature that resists early release.

Analysis of Recombinant Adenovirus Vectors by Ion Trap Charge Detection Mass Spectrometry: Accurate Molecular Weight Measurements beyond 150 MDa.

Adenovirus is one of the largest nonenveloped, double-stranded DNA viruses. It is widely used as a gene therapy vector and has recently received a lot of attention as a novel vaccine platform for SARS-CoV-2. Human adenovirus 5 (HAdV5) contains over 2500 protein molecules and has a 36 kbp genome. Adenovirus is well beyond the range of conventional mass spectrometry, and it was unclear how well such a large complex could be desolvated. Here, we report molecular weight (MW) distributions measured for HAdV5 and for 11 recombinant AdV vectors with genomes of varying lengths. The MW distributions were recorded using ion trap charge detection mass spectrometry (CDMS), a single-particle technique where m/z and charge are measured for individual ions. The results show that ions as large as 150 MDa can be effectively desolvated and accurate MW distributions obtained. The MW distribution for HAdV5 contains a narrow peak at 156.1 MDa, assigned to the infectious virus. A smaller peak at 129.6 MDa is attributed to incomplete particles that have not packaged a genome. The ions in the 129.6 MDa peak have a much lower average charge than those in the peak at 156.1 MDa. This is attributed to the empty particles missing some or all of the fibers that decorate the surface of the virion. The MW measured for the mature virus (156.1 MDa) is much larger than that predicted from sequence masses and copy numbers of the constituents (142.5 MDa). Measurements performed for recombinant AdV as a function of genome length show that for every 1 MDa increase in the genome MW, the MW of the mature virus increases by around 2.3 MDa. The additional 1.3 MDa is attributed to core proteins that are copackaged with the DNA. This observation suggests that the discrepancy between the measured and expected MWs for mature HAdV5 is due to an underestimate in the copy numbers of the core proteins.

Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer.

We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. To determine a possible reason for this outcome, we characterized the proportion and composition of the empty and packaged capsids. Both analytical ultracentrifugation (AUC) and differential centrifugation sedimentation (DCS, a rapid and quantitative method for measuring adenoviral packaging variants) were employed for an initial assessment of genome packaging and showed multiple species whose abundance deviated between the virus builds but not manufacturing campaigns. Identity of the packaging variants was confirmed by charge detection mass spectrometry (CDMS), the first known application of this technique to analyze adenovirus. The empty and packaged capsid populations were separated via preparative ultracentrifugation and then combined into a series of mixtures. These mixtures showed the oft-utilized denaturing A260 adenoviral particle titer method will underestimate the actual particle titer by as much as three-fold depending on the empty/full ratio. In contrast, liquid chromatography with fluorescence detection proves to be a superior viral particle titer methodology.

Quantitative analysis of genome packaging in recombinant AAV vectors by charge detection mass spectrometry.

Recombinant adeno-associated virus (rAAV) has emerged as an important gene therapy vector with many clinical trials currently in progress. Analytical characterization and quantitation of particle content remain challenges in both the development and production of rAAV vectors. In this study, charge detection mass spectrometry (CDMS) and gel electrophoresis are used to characterize the DNA content of recombinant AAV8 (rAAV8) vectors with a wide range of target genome sizes. We show that the differences between the masses of empty particles and particles with the genome of interest (GOI) are correlated with the expected genome mass. A small systematic deviation (around 2%) is attributed to the packaging of counterions along with the DNA. In addition to the GOI, a broad distribution of heterogeneous DNA is packaged. The distribution peaks are close to the packaging capacity of the rAAV8 vectors. There is also evidence for the co-packaging of small DNA fragments along with the GOI. Finally, we present evidence that incubation at an elevated temperature can reduce the heterogeneity of the packaged DNA. Taken together, these results show that CDMS is a viable tool for characterization of the packaged genome.

Small- and large-sized iron(II, III) oxide nanoparticles for surface-assisted laser desorption/ionization mass spectrometry of small biomolecules.

RATIONALE: Common surface-assisted laser desorption/ionization (SALDI) surfaces are functionalized to improve mass spectrometric detection. Such surfaces are selective to certain group(s) of compounds. The application of universal and sensitive SALDI surfaces with appropriate size/surface area is paramount. In this study, two different sizes/surface areas of Fe3 O4 are compared as SALDI surfaces. METHODS: For accurate surface area comparisons, the physical properties of the Fe3 O4 nanoparticles used as SALDI surfaces were determined using scanning electron microscopy, X-ray diffractometry, and N2 Brunauer-Emmet-Teller adsorption techniques. SALDI mass spectrometry (MS) data were acquired using a time-of-flight (TOF) mass spectrometer operated in the linear mode and equipped with a 50-Hz pulsed nitrogen laser (at 337 nm). Small biomolecules (adenosine, glucose, sucrose, tryptophan, and tripeptide) and a real sample (human serum) were analyzed. RESULTS: The average sizes/specific surface areas of the SALDI surfaces of the small- and large-sized Fe3 O4 nanoparticles were ~21 nm/~82 m2 /g and ~39 nm/~38 m2 /g, respectively. An overall ~2.0-fold enhancement in signal-to-noise ratios was observed for the ionic species of the analyzed biomolecules in SALDI-MS using small-sized Fe3 O4 in comparison to large-sized Fe3 O4 nanoparticles. MS sensitivity from adenosine calibration curves (concentration between 0.05 and 10.0 mM) was ~2.0-fold higher for small-sized than large-sized Fe3 O4 nanoparticles as SALDI surfaces. CONCLUSIONS: We have shown that transition-metal oxides such as Fe3 O4 nanoparticles are suitable and efficient surfaces for SALDI-TOF-MS analysis of small biomolecules. We observed improvement in signal-to-noise ratios and detection sensitivity for the analyzed samples from SALDI surfaces using small-sized (possessing larger surface area) than large-sized Fe3 O4 nanoparticles.