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

Abstract

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.

Keywords: AAV; CDMS; GOI; adeno-associated virus; charge detection mass spectrometry; genome; mass spectrometry; rAAV.

Graphical abstract

Figure 1

CDMS measurements for empty AAV8 and AAV8 with genomes (A) Mass distribution for empty AAV8. The inset shows an expanded view of the main peak. The orange line shows the mass distribution calculated for a 1:1:10 ratio of VP1–VP3 considering the resolution for a trapping time of 100 ms. (B) Charge versus mass scatterplot for empty AAV8. (C) Mass distributions measured for AAV8 particles with the genomes in Table 1. The dashed blue lines show the expected masses for particles with a single genome, and the dashed green lines show the expected masses for two packaged genomes. The red dashed line at 5.33 MDa shows what appears to be the approximate packaging capacity of AAV8. In all cases, a bin width of 20 kDa was employed and all spectra contain around 10,000 ions.

Figure 2

Charge versus mass scatterplots for representative genomes (A) CMV-CRE. (B) CAG-GFP. (C) CBA-GFP.

Figure 3

Alkaline gel measurements of ssDNA from AAV8 vectors Two measurements are shown for each genome in Table 1: −, without DNase I; +, with DNase I; L, ladder. GOI lengths are shown in parentheses.

Figure 4

TapeStation measurements of dsDNA from AAV8 vectors Three measurements are shown for each genome in Table 1: −, without DNase I; +, with DNase I; +∗, with DNase I and purified (see Materials and methods for details). L, ladder; W, water. The green band at the bottom of the gel indicates the internal marker at 100 bp. GOI lengths are shown in parentheses.

Figure 5

Incubation of AAV8 particles with EF1a-GFP genome The CDMS mass distribution measured after incubation at 65°C for 15 min is shown in blue. The distribution measured before incubation is shown in green for comparison. The orange line shows the expected peak width considering the capsid heterogeneity and instrumental resolution.

Figure 6

The measured DNA masses plotted against the DNA sequence masses The measured masses were obtained from the difference between the masses measured for the full and empty particles and the sequence masses are from Table 1. The red points are the experimental data. The dashed black line shows the expected behavior when the measured DNA mass equals the expected mass (slope = 1.0). The red line shows a least-squares fit to the data points. The slope is 1.041, the intercept is −0.016 MDa, and R² is 0.9956.

Figure 7

CDMS measurements for ATCC AAV8 reference standard (A) plot of the mass distribution (blue line). The yellow line shows the spectrum ×10 and offset vertically. The dashed red line shows the distribution expected when the capsid heterogeneity and experimental resolution are included. (B) charge versus mass scatterplot where the measurement for each ion is represented by a point.