Seitz Immanuel P, Rodríguez-Bocanegra Eduardo, Bucher Kirsten, Reichel Felix F, Michalakis Stylianos, Romanovsky Dimitri, Biel Martin, Wissinger Bernd, Bartz-Schmidt Karl-Ulrich, Peters Tobias, Fischer Manuel D
Centre for Ophthalmology, University Hospital Tübingen, Tübingen, Germany.
Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom.
Invest Ophthalmol Vis Sci. 2025 Jun 2;66(6):38. doi: 10.1167/iovs.66.6.38.
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.
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.
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).
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.
用于基因治疗的腺相关病毒（AAV）制剂含有与生产相关的杂质，如残留宿主细胞蛋白（HCP）。本研究的目的是调查AAV制剂中高水平的HCP是否与炎症增加和眼部耐受性降低有关。
分析三批临床级AAV8载体中与生产相关杂质的存在情况。使用质谱对这些杂质的HCP成分进行表征。然后比较各批次在原代人浆细胞样树突状细胞（pDCs）和THP-1细胞中诱导细胞因子反应的能力。此外，对健康非人灵长类动物的眼部安全性研究结果进行事后分析，以调查HCP水平对炎症临床体征和脉络膜视网膜萎缩（CRA）发展的影响。
载体批次的HCP水平显示出高达约40倍的差异。人半乳糖凝集素-3结合蛋白是唯一主要的HCP污染物。用高HCP批次刺激人pDCs和THP-1细胞并未导致细胞因子反应增加。高HCP也未加剧炎症的临床体征。然而，在视网膜成像中，高HCP处理组眼睛的CRA病变明显更大（P = 0.001 - 0.048）。
HCP杂质的复杂性较低，但各批次之间其丰度存在明显差异。高HCP水平在体内和体外均未表现出明显的免疫原性。然而，尽管存在统计局限性，但它们似乎与CRA增加有关。因此，不能排除高HCP水平对视网膜耐受性产生负面影响的可能性。
