Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components
Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components
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DOI：
10.1016/j.vaccine.2019.04.056
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发表时间：
2019-11-08
期刊：
影响因子：
5.5
通讯作者：
Douglas, Alexander D.
中科院分区：
文献类型：
--
作者：
Fedosyuk, Sofiya;Merritt, Thomas;Douglas, Alexander D.
已经报道了用于生产非复制型腺病毒载体的各种符合良好生产规范（GMP）的方法，但是仍然存在重要的挑战。目前大多数腺病毒载体的临床开发使用猿猴腺病毒或罕见的人血清型，而报道的生产工艺主要使用血清型如AdHu 5，其与临床疫苗开发的相关性值得怀疑。许多临床相关的疫苗转基因干扰腺病毒复制，而大多数报道的工艺开发使用选择的抗原或甚至模型转基因，如荧光蛋白，其几乎不引起这种干扰。需要用于早期疫苗试验的非复制型腺病毒载体的小GMP批次的快速生产平台，特别是在准备应对新出现的病原体爆发时。这样的平台必须对转基因的变异具有鲁棒性，并且理想地还能够产生多于一种血清型的腺病毒。这也是非常可取的，这样的过程是很容易在新的设施中使用市售的一次性使用的材料，避免了定制的工具或清洁验证的需要，并为他们很容易扩展为后期阶段的研究。在这里，我们报告了这样一个过程的发展，使用一次性搅拌罐生物反应器，转基因抑制HEK 293细胞启动子组合，以及完全一次性使用的过滤和离子交换组件。我们证明了该方法对狂犬病、疟疾和裂谷热候选疫苗的适用性，每种候选疫苗都基于不同的腺病毒血清型。我们比较了一系列市售离子交换介质的性能，包括我们认为是首次公开使用的用于腺病毒纯化的新型介质（NatriFlo（R）HD-Q Merck）。我们证明了每种疫苗需要最小的工艺个性化，并且该产品符合监管质量预期。细胞特异性产率处于文献中先前报道的上限，体积产率在每升培养物1 × 10（13）- 5 × 10（13）个纯化病毒颗粒的范围内，因此2-4 L工艺足以生产用于早期试验的疫苗。该工艺易于转移至任何具有哺乳动物细胞培养和无菌产品无菌灌装能力的GMP设施。(C)2019年，任作家。爱思唯尔有限公司出版
A variety of Good Manufacturing Practice (GMP) compliant processes have been reported for production of non-replicating adenovirus vectors, but important challenges remain. Most clinical development of adenovirus vectors now uses simian adenoviruses or rare human serotypes, whereas reported manufacturing processes mainly use serotypes such as AdHu5 which are of questionable relevance for clinical vaccine development. Many clinically relevant vaccine transgenes interfere with adenovirus replication, whereas most reported process development uses selected antigens or even model transgenes such as fluorescent proteins which cause little such interference. Processes are typically developed for a single adenovirus serotype - transgene combination, requiring extensive further optimization for each new vaccine.There is a need for rapid production platforms for small GMP batches of non-replicating adenovirus vectors for early-phase vaccine trials, particularly in preparation for response to emerging pathogen outbreaks. Such platforms must be robust to variation in the transgene, and ideally also capable of producing adenoviruses of more than one serotype. It is also highly desirable for such processes to be readily implemented in new facilities using commercially available single-use materials, avoiding the need for development of bespoke tools or cleaning validation, and for them to be readily scalable for later-stage studies.Here we report the development of such a process, using single-use stirred-tank bioreactors, a transgene-repressing HEK293 cell - promoter combination, and fully single-use filtration and ion exchange components. We demonstrate applicability of the process to candidate vaccines against rabies, malaria and Rift Valley fever, each based on a different adenovirus serotype. We compare performance of a range of commercially available ion exchange media, including what we believe to be the first published use of a novel media for adenovirus purification (NatriFlo (R) HD-Q Merck). We demonstrate the need for minimal process individualization for each vaccine, and that the product fulfils regulatory quality expectations. Cell-specific yields are at the upper end of those previously reported in the literature, and volumetric yields are in the range 1 x 10(13) - 5 x 10(13) purified virus particles per litre of culture, such that a 2-4 L process is comfortably adequate to produce vaccine for early-phase trials. The process is readily transferable to any GMP facility with the capability for mammalian cell culture and aseptic filling of sterile products. (C) 2019 The Authors. Published by Elsevier Ltd.