Leibiger Thomas M, Min Lie, Lee Kelvin H
University of Delaware, Department of Chemical and Biomolecular Engineering, Newark, DE 19713, USA.
Mol Ther Methods Clin Dev. 2024 Nov 18;32(4):101383. doi: 10.1016/j.omtm.2024.101383. eCollection 2024 Dec 12.
To better understand host cell protein (HCP) retention in adeno-associated virus (AAV) downstream processes, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) was used to quantitatively profile residual HCPs for four AAV serotypes (AAV2, -5, -8, and -9) produced with HEK293 cells and purified using POROS CaptureSelect AAVX affinity chromatography. A broad range of residual HCPs were detected in affinity eluates after purification ( = 2,746), and HCP profiles showed universally present species ( = 1,117) and species unique to one or more AAV serotype. SWATH-MS revealed that HCP persistence was dominated by high-abundance conserved species (HACS), which appeared across all serotype conditions studied. Due to the notable contribution of these species to overall residual HCP levels, physical and functional characteristics of HACS were examined to determine trends that coincide with persistence. Subnetwork interaction mapping and Gene Ontology function enrichment analysis revealed extensive physical interactions between these proteins and significant enrichment for biological processes, molecular functions, and reactome pathways related to protein folding, nucleic acid binding, and cellular stress. The abundant and conserved nature of these HCPs and their functions offers a new perspective for mechanistic evaluations of impurity retention for AAV downstream processes.
为了更好地理解腺相关病毒（AAV）下游工艺中宿主细胞蛋白（HCP）的残留情况，采用了所有理论碎片离子质谱的连续窗口采集（SWATH-MS）技术，对用HEK293细胞生产并使用POROS CaptureSelect AAVX亲和色谱法纯化的四种AAV血清型（AAV2、-5、-8和-9）的残留HCP进行定量分析。纯化后的亲和洗脱液中检测到了广泛的残留HCP（n = 2,746），HCP图谱显示存在普遍存在的物种（n = 1,117）以及一种或多种AAV血清型特有的物种。SWATH-MS显示，HCP的残留主要由高丰度保守物种（HACS）主导，这些物种在所研究的所有血清型条件下均出现。由于这些物种对总体残留HCP水平有显著贡献，因此对HACS的物理和功能特性进行了研究，以确定与残留相关的趋势。子网相互作用图谱和基因本体功能富集分析揭示了这些蛋白质之间广泛的物理相互作用，以及与蛋白质折叠、核酸结合和细胞应激相关的生物过程、分子功能和反应组途径的显著富集。这些HCP的丰富性、保守性及其功能为AAV下游工艺中杂质残留的机制评估提供了新的视角。
