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

Abstract

Different manufacturing approaches have been used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Sf9 insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed for the first time a highly controlled comparative production analysis varying only the host cell species but keeping all other rAAV production parameters the same. We demonstrate that host cell species is critical for determining vector potency. Given these key findings, we then sought to deeply characterize differences in rAAVs when produced by these two manufacturing platforms with multiple analytical approaches including: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM, denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and comparative functional transduction assessments in vitro and in vivo, including in humanized liver mice. Using these tools weve made two major discoveries: 1) rAAV capsids have post-translational modifications (PTMs) including glycosylation, acetylation, phosphorylation, methylation and deamidation, and these PTMs differ between platforms; 2) rAAV genomes are methylated during production, and these methylation marks are also differentially deposited between platforms. In addition, our data also demonstrate that host cell protein impurities differ between platforms and can have their own PTMs including potentially immunogenic N-linked glycans. We show that human-produced rAAVs are more potent than baculovirus-Sf9 vectors in various cell types in vitro (P < 0.05-0.0001), in various mouse tissues in vivo (P < 0.03-0.0001), and in human liver in vivo (P < 0.005). Collectively, our findings were reproducible across vendors, including commercial manufacturers, academic core facilities, and individual laboratory preparations. These vector differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity as well as cost considerations.