Figure 1.

AAV bioprocessing and analysis

Schematic overview of rAAV production in either HEK293 or SF9 host cells for a designed transgene of around 2.6 kb. After transfection of (or infection with) the corresponding transgene (1) (if replicated properly) they will yield an encapsulated 800-kDa genome (2). Other encapsulated off-target genomes could originate from self-complementary ssDNA dimeric variants (3) as well as truncated genomes (4). These gene products are all encapsulated (5) and will yield a mixture of empty and (partly) filled particles. 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. rAAV capsids show additionally a wide distribution of empty, partially filled, and filled capsids, requiring additional purification and monitoring. Detailed characterization is important as only a small number of the infectious particles will transduce their genome. This efficiency is sensitive to the presence of empty capsids and capsids filled with off-target genomes. For monitoring this, AUC and EM are the industry standards, whereby the former can also serve as preparative method. Here, Orbitrap-based CD-MS is explored for quality control, as it is sensitive, facile, and can yield information on capsid/genome integrity.