Methods Matter -- Standard Production Platforms For Recombinant AAV Can Produce Chemically And Functionally Distinct Vectors

Different manufacturing approaches have been used in the production of recombinant adeno-associated virus (rAAV). We sought to characterize differences in rAAV vectors when produced by the two leading manufacturing platforms; live baculovirus infection of Sf9 insect cells and transiently-transfected human HEK293 cells. We used multiple analytical approaches, including proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM, denaturation assays, next-generation sequencing of packaged genomes, human cytokine profiling in response to vector transduction, and comparative functional transduction assessments in vivo and in vitro. Our data support the following findings: 1) rAAV capsids have post-translational modifications (PTMs); 2) vector lot modifications included glycosylation, acetylation, phosphorylation, methylation and deamidation; 3) capsid PTMs differ when produced in either platform; 4) impurities were different in vectors when produced in either platform; 5) impurities can also have their own PTMs, including N-linked glycans; 6) capsid PTMs and impurities were seen across all rAAV serotypes, manufacturers, and purification types; 7) there was no difference in the packaged rAAV genome sequence in either platform; 8) baculovirus-Sf9 vector lots have insect and baculoviral impurities and can have poorer packaging percentages than human-produced vector; 9) rAAV capsids have no significant structural differences when produced in either platform; 10) when given at the same vector genome dose, human produced rAAVs can be more potent than baculovirus-Sf9 vectors in vitro and in vivo (P