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Rational downstream development for adeno-associated virus full/empty capsid separation – A streamlined methodology based on high-throughput screening and mechanistic modeling

下游加工 衣壳 化学 色谱法 洗脱 高通量筛选 腺相关病毒 重组DNA 生物化学 载体(分子生物学) 基因
作者
William R. Keller,Angela L. Picciano,Kelly G. Wilson,Jingfen Xu,Harshit Khasa,Michaela Wendeler
出处
期刊:Journal of Chromatography A [Elsevier]
卷期号:1716: 464632-464632
标识
DOI:10.1016/j.chroma.2024.464632
摘要

Recombinant adeno-associated virus (AAV) has emerged as one of the most promising systems for therapeutic gene delivery and has demonstrated clinical success in a wide range of genetic disorders. However, manufacturing of high-quality AAV in large amounts still remains a challenge. A significant difficulty for downstream processing is the need to remove empty capsids that are generated in all currently utilized expression systems and that represent product-related impurities that adversely affect safety and efficacy of AAV vectors. Empty and full capsids exhibit only subtle differences in surface charge and size, making chromatography-based separations highly challenging. Here, we present a rapid methodology for the systematic process development of the crucial AAV full/empty capsid separation on ion-exchange media based on high-throughput screening and mechanistic modeling. Two of the most commonly employed serotypes, AAV8 and AAV9, are used as case studies. First, high-throughput studies in filter-plate format are performed that allow the rapid and comprehensive study of binding and elution behavior of AAV on different resins, using different buffer systems, pH, salt conditions, and solution additives. Small amounts of separated empty and full AAV capsids are generated by iodixanol gradient centrifugation that allow studying the binding and elution behavior of the two vector species separately in miniaturized format. Process conditions that result in maximum differences in elution behavior between empty and full capsids are then transferred to benchtop chromatography systems that are used to generate calibration data for the estimation of steric mass-action isotherm and mass transport parameters for process simulation. The resulting column models are employed for in-silico process development that serves to enhance understanding of separation constraints and to identify optimized conditions for the removal of empty particles. Finally, optimized separation conditions are verified experimentally. The methodology presented in this work provides a systematic framework that affords mechanistic understanding of the crucial empty/full capsid separation and accelerates the development of a scalable AAV downstream process.
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