Bláha Benjamin A F, Morris Stephen A, Ogonah Olotu W, Maucourant Sophie, Crescente Vincenzo, Rosenberg William, Mukhopadhyay Tarit K
The Advanced Centre for Biochemical Engineering, Dept. of Biochemical Engineering, University College London, London, WC1E 7JE, U.K.
iQur Limited, London Bioscience Innovation Centre, London, NW1 0NH, U.K.
Biotechnol Prog. 2018 Jan;34(1):130-140. doi: 10.1002/btpr.2555. Epub 2017 Oct 16.
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. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130-140, 2018.
小型发酵平台的时间和成本效益只有通过采用有助于在小样本量下实现高通量的互补技术才能获得。微生物细胞破碎是实验通量的主要瓶颈，并且通常限于大的处理量。此外，对于像巴斯德毕赤酵母这样的坚硬酵母物种，不存在有效的高通量破碎方法。本文描述了一种基于自适应聚焦声学（AFA）的自动化、小型化、高通量、非接触、可扩展平台的开发，该平台用于破碎巴斯德毕赤酵母并回收细胞内异源蛋白。通过研究容器设计和一个新的酶预处理步骤建立了增强的AFA模式。研究了三种不同的AFA模式，并与高压匀浆的性能进行了比较。对于这些细胞破碎模式中的每一种，都开发了响应模型以考虑五个不同的性能标准。使用多个响应不仅表明不同的响应最优值需要不同的操作参数，最高的产品纯度需要其他标准的次优值，而且还允许基于AFA的方法模拟大规模匀浆过程。这些结果表明，AFA介导的细胞破碎可用于广泛的应用，包括缓冲液开发、菌株选择、发酵工艺开发和整个生物工艺整合。©2017美国化学工程师学会生物技术进展，34:130 - 140，2018。
