Investigating the Immunogenic Potential of Variations in Host Cell Protein Levels in Clinical-Grade AAV8 Products.

PURPOSE: Adeno-associated virus (AAV) formulations for gene therapy contain manufacturing-associated impurities such as residual host cell protein (HCP). The aim of this study was to investigate whether high levels of HCP in AAV formulations are associated with increased inflammation and reduced ocular tolerability. METHODS: Three lots of clinical-grade AAV8 vector were analyzed for the presence of manufacturing-associated impurities. The HCP component of these impurities was characterized using mass spectrometry. Lots were then compared regarding their capacity to induce a cytokine response in primary human plasmacytoid dendritic cells (pDCs) and THP-1 cells. Furthermore, the results of an ocular safety study in healthy nonhuman primates were analyzed post hoc to investigate the influence of HCP levels on clinical signs of inflammation and chorioretinal atrophy (CRA) development. RESULTS: Vector lots displayed up to a ∼40-fold variation in HCP levels. Human galactin-3-binding protein was the only major HCP contaminant. Stimulation of human pDCs and THP-1 cells with a high HCP lot did not result in an increased cytokine response. High HCP also did not exacerbate clinical signs of inflammation. However, on retinal imaging, CRA lesions were significantly larger in high HCP-treated eyes (P = 0.001-0.048). CONCLUSIONS: HCP impurities were of low complexity, but pronounced variations in their abundance were observed between lots. High HCP levels were not overtly immunogenic in vivo and in vitro. However, despite statistical limitations, they seemed to be associated with increased CRA. Thus, a negative effect of high HCP levels on retinal tolerability could not be ruled out.