Abstract
Adeno-associated viruses (AAVs) represent important gene therapy vectors with several approved clinical applications and numerous more in clinical trials. 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. 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. Here we show that Orbitrap-based charge-detection mass spectrometry allows the very sensitive quantification of all these different AAV bioprocessing products. 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 ∼1013 viral capsids per milliliter. 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.
Highlights
Adeno-associated viruses (AAVs) are small viruses with a singlestranded DNA genome that is encapsidated by a stochastic mixture of 60 VP1, VP2, or VP3.1,2 The virus belongs to the genus Dependoparvovirus, and is dependent on coinfection with helper viruses like adeno- or herpesviruses
Optimizing the analysis of co-occurring AAV particles in charge-detection mass spectrometry (CD-MS) Here we aimed to quantitatively measure co-occurring recombinant AAVs (rAAVs) particles ranging in mass from about 3.6 to 5.3 MDa
RAAV particles are directly diluted, from their storage solution, into aqueous ammonium acetate and introduced into a mass spectrometer where they are ionized by electrospray ionization under native conditions.[26]
Summary
Adeno-associated viruses (AAVs) are small viruses with a singlestranded DNA (ssDNA) genome that is encapsidated by a stochastic mixture of 60 VP1, VP2, or VP3.1,2 The virus belongs to the genus Dependoparvovirus, and is dependent on coinfection with helper viruses like adeno- or herpesviruses. For gene-delivery purposes, the natural AAV genome is replaced with a transgene, flanked by the natural AAV inverted terminal repeats (ITRs). These recombinant AAVs (rAAVs) are typically produced in dedicated host expression systems where the transgene, the structural AAV genes, and the required helper genes are co-transfected or stably integrated.[4,5,6,7] Human HEK293 or Sf9 insect cells are currently the most often used expression systems used for expression of AAV, other and/or adapted cell lines have been explored as well.[8]. This diversity of products, combined with the poor scalability of the used expression systems, makes AAV production and purification challenging and expensive
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
