All measured masses are summarized in Table 1. The two most prominent particle populations represent the empty (∼23,000 particles, MW = 3.85 MDa) and genome-filled rAAV6a particles (∼40,400 particles, MW = 4.63 MDa), with a relative mass difference of ∼800 kDa, due to the packaging of the expected genome. From 2D CD-MS, two prominent co-occurring distributions can be observed, but also two weaker particle populations, all with distinct masses. Cumulatively, the 2D CD-MS spectrum shown in Figure 2A shows the data for about ∼76,000 individual rAAV6a particles. By CD-MS, the obtained two-dimensional (2D) histogram and the corresponding mass histogram for an AAV6a serotype, expressed with the above-mentioned transgene, are shown in Figures 2A and 2B. Besides packing this genome, other possible genome variants that may be packed by the AAV are a self-complementary ssDNA (MW ∼2 × 800 kDa) and truncated forms of the genome as further depicted in Figure 1. The analyzed particles were expressed with a common expression cassette with an approximately 2.5-kb long genome (with a molecular weight of ∼800 kDa). The importance of using this optimized acquisition approach becomes most evident when aiming to analyze and quantify low-abundant encapsulated genome variants, as demonstrated in Figure 2. Results Optimizing the analysis of co-occurring AAV particles in CD-MS This new approach can contribute to the optimization of the bioprocessing of rAAVs, producing more particles containing the desired intact transgene. We show that the quantitative accuracy of the CD-MS method to distinguish filled from empty particles is in the range of 1%–3% (see Table S1). We introduce an improved acquisition method with better signal utilization that can accurately and reproducible detect rAAV particle populations as low as 2%, also identifying some less-abundant, likely scAAV, variants as off-target products in rAAV preparations. We determine the mass and abundance of various co-occurring rAAV particles in less than 30 min, using only 1 μL of sample at typical AAV working concentrations (2 × 10 13 vc/mL). Here we present an optimized workflow enabling the mass analysis of heterogeneous rAAV particles, whereby the mass resolving power and accuracy attainable allow us to determine and quantify genome integrity. Here, Orbitrap-based CD-MS is explored for quality control, as it is sensitive, facile, and can yield information on capsid/genome integrity. For monitoring this, AUC and EM are the industry standards, whereby the former can also serve as preparative method. This efficiency is sensitive to the presence of empty capsids and capsids filled with off-target genomes. Detailed characterization is important as only a small number of the infectious particles will transduce their genome. rAAV capsids show additionally a wide distribution of empty, partially filled, and filled capsids, requiring additional purification and monitoring. The initial number of other contaminants (DNA, side product, host cell proteins) can be several orders of magnitude higher than that of rAAV capsids and require extensive purification. These gene products are all encapsulated (5) and will yield a mixture of empty and (partly) filled particles. Other encapsulated off-target genomes could originate from self-complementary ssDNA dimeric variants (3) as well as truncated genomes (4). After transfection of (or infection with) the corresponding transgene (1) (if replicated properly) they will yield an encapsulated 800-kDa genome (2). Schematic overview of rAAV production in either HEK293 or SF9 host cells for a designed transgene of around 2.6 kb. The method quickly assesses the integrity and amount of genome packed AAV particles to support AAV bioprocessing and characterization of this rapidly emerging class of advanced drug therapies. A protocol is presented that allows the quantification of genome-packed AAV preparations in under half an hour, requiring only micro-liter quantities of typical AAV preparations with ∼10 13 viral capsids per milliliter. Here we show that Orbitrap-based charge-detection mass spectrometry allows the very sensitive quantification of all these different AAV bioprocessing products. Current methods to monitor genome packaging have limited sensitivity, a high demand on labor, and struggle to distinguish between packaging of the intended genome or unwanted side-products. Genome packaging is an essential step in the bioprocessing of AAVs and needs to be tightly monitored to ensure the proper delivery of transgenes and the production of effective drugs.
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