Microscale Perfusion‐Based Cultivation for Pichia pastoris Clone Screening Enables Accelerated and Optimized Recombinant Protein Production Processes,Biotechnology Journal

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Microscale Perfusion‐Based Cultivation for Pichia pastoris Clone Screening Enables Accelerated and Optimized Recombinant Protein Production Processes
Biotechnology Journal ( IF 3.2 ) Pub Date : 2020-09-15 , DOI: 10.1002/biot.202000215
Damiano Totaro _{1,

2,

3} , Bojana Radoman _{1,

2} , Bernhard Schmelzer _{1,

2} , Mario Rothbauer ₃ , Matthias G Steiger _{1,

2,

4} , Torsten Mayr ₅ , Michael Sauer _{1,

2} , Peter Ertl ₃ , Diethard Mattanovich _{1,

2}

Affiliation

Pichia pastoris has emerged in the past years as a promising host for recombinant protein and biopharmaceutical production. In the establishment of high cell density fed‐batch biomanufacturing, screening phase and early bioprocess development (based on microplates and shake flasks) still represent a bottleneck due to high‐cost and time‐consuming procedures as well as low experiment complexity. In the present work, a screening protocol developed for P. pastoris clone selection is implemented in a multiplexed microfluidic device with 15 μL cultivation chambers able to operate in perfusion mode and monitor dissolved oxygen content in the culture in a non‐invasive way. The setup allowed us to establish carbon‐limited conditions and evaluate strain responses to different input variables. Results from micro‐scale perfusion cultures are then compared with 1L fed‐batch fermentation. The best producer in terms of titer and productivity is rapidly identified after 12 h from inoculation and the results confirmed by lab‐scale fermentation. Moreover, the physiological analyses of the strains under different conditions suggested how more complex experimental conditions are achievable despite the relatively easy, straight‐forward, and cost‐effective experimental setup. Implementation and standardization of these micro‐scale protocols could reduce the demand for lab‐scale bioreactor cultivations thus accelerating the development of protein production processes.

中文翻译：

基于微量灌流的巴斯德毕赤酵母克隆培养能够加速和优化重组蛋白的生产过程

在过去的几年中，巴斯德毕赤酵母已经成为重组蛋白和生物制药生产的有希望的宿主。在建立高细胞密度的分批生物生产过程中，筛选阶段和早期生物工艺开发（基于微孔板和摇瓶）仍然存在瓶颈，原因是过程成本高昂，耗时且实验复杂度低。在目前的工作中，为P. pastoris开发了一种筛选方案克隆选择是在具有15μL培养箱的多路微流控设备中实施的，该培养箱能够以灌注模式运行并以非侵入性方式监控培养物中的溶解氧含量。该设置使我们能够建立碳限制条件，并评估应变对不同输入变量的响应。然后将微量灌流培养的结果与1L分批补料发酵进行比较。接种后12 h，可以快速确定效价和生产率方面的最佳生产者，实验室规模的发酵证实了该结果。此外，在不同条件下对菌株进行的生理分析表明，尽管相对简单，直接且具有成本效益的实验设置，但如何能够实现更复杂的实验条件。

更新日期：2020-09-15

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