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Development of a high-throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design

机译：快速生物过程设计中Pichia Pastoris的高通量微观细胞中断平台的开发

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The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high-throughput disruption methods exist. The development of an automated, miniaturized, high-throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high-pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. (c) 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130-140, 2018
机译：小型化发酵平台的时间和成本益处只能通过采用促进在小样本量的高通量的互补技术来获得。微生物细胞破坏是实验过程中的主要瓶颈，并且通常仅限于大加工量。此外，对于刚性酵母物种，例如毕赤酵母，不存在有效的高通量破坏方法。基于自适应聚焦声学（AFA）的自动化，小型化，高通量，非接触，可伸缩平台的发展，以破坏P.牧场和恢复细胞内异源蛋白质。通过研究血管设计和新的酶预处理步骤，建立了AFA的增强模式。研究了三种不同的AFA模式，并与性能高压均质化进行比较。对于这些细胞中断模式中的每种模式，开发了响应模型以考虑五种不同的性能标准。使用多个响应不仅表明不同的响应Optima需要不同的操作参数，具有最高的产品纯度，需要用于其他标准的次优值，但也允许基于AFA的方法模仿大规模均质化过程。这些结果表明，AFA介导的细胞破坏可用于各种应用，包括缓冲区开发，应变选择，发酵过程开发和整个生物过程集成。 （c）2017美国化学工程师生物科技学院。 Prog。，34：130-140,2018

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