Adenovirus 5 recovery using nanofiber ion-exchange adsorbents.
Adenovirus 5 recovery using nanofiber ion-exchange adsorbents.
复制标题
使用纳米纤维离子交换吸附剂回收腺病毒 5。
DOI：
10.1002/bit.26972
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发表时间：
2019
影响因子：
3.8
通讯作者：
Turnbull J
中科院分区：
文献类型：
--
作者：
Turnbull J
腺病毒等病毒载体在疫苗和基因治疗方面有成功的应用，但高质量病毒的制造仍然是一个挑战。人们希望使用基于吸附的层析分离，从而有效地支持治疗性蛋白质的制造。然而，这类产品在尺寸和稳定性方面的根本差异意味着有必要重新审视吸附剂的形态和表面化学的设计。在这项研究中，用定义密度的季胺配体衍生的纤维素纳米纤维离子交换吸附剂的行为被表征以解决这一问题。这种材料之所以被选中，是因为它具有大的病毒颗粒可接近的表面积和快速的处理时间。首先，研究了低(440 微克分子/克)、中(750微克分子/克)和高(1029微克分子/克)配基密度下表面化学对感染产物回收的影响。在较高密度下，产物的稳定性降低，这种影响随24 分钟的吸附时间延长而增加，在低配基密度下仅损失约10%，而在高配基密度下损失约50%。这可以通过使用高流速将循环时间减少到~1 分钟来缓解。其次，考察了配基密度对分离分辨率的影响。了解病毒质量的关键是病毒颗粒：传染性病毒颗粒的比率。研究发现，这一参数可以通过配基密度和洗脱策略来控制。综上所述，这为病毒载体的分离提供了基础，通过使用高液体速度将负载和柱上时间降至最低，同时分离关键产品和与工艺相关的杂质，从而实现了病毒载体的低滴度和不稳定性质。
Viral vectors such as adenovirus have successful applications in vaccines and gene therapy but the manufacture of the high‐quality virus remains a challenge. It is desirable to use the adsorption‐based chromatographic separations that so effectively underpin the therapeutic protein manufacture. However fundamental differences in the size and stability of this class of product mean it is necessary to revisit the design of sorbent's morphology and surface chemistry. In this study, the behaviour of a cellulose nanofiber ion‐exchange sorbent derivatised with quaternary amine ligands at defined densities is characterised to address this. This material was selected as it has a large accessible surface area for viral particles and rapid process times. Initially, the impact of surface chemistry on infective product recovery using low (440 µmol/g), medium (750 µmol/g), and high (1029 µmol/g) ligand densities is studied. At higher densities product stability is reduced, this effect increased with prolonged adsorption durations of 24 min with just ~10% loss at low ligand density versus ~50% at high. This could be mitigated by using a high flow rate to reduce the cycle time to ~1 min. Next, the impact of ligand density on the separation's resolution was evaluated. Key to understanding virus quality is the virus particle: infectious virus particle ratio. It was found this parameter could be manipulated using ligand density and elution strategy. Together this provides a basis for viral vector separations that allows for their typically low titres and labile nature by using high liquid velocity to minimise both load and on‐column times while separating key product and process‐related impurities.