SNM: Scalable Cell-free Protein Manufacturing via NanoClay-DNA (NanoCD) Microdonuts

蛋白质正在成为治疗和预防疾病的重要药物。事实上，在目前世界上十大最畅销的药物中，有八种是基于蛋白质的。目前，几乎所有的蛋白质都是由生物体制造的。不幸的是，使用活生物体生产蛋白质具有各种限制和问题，包括低生产率和高设置成本。最严重的是，生物体需要得到足够的喂养和良好的照顾，这在工业规模上并不容易。在这个项目中，由康奈尔大学Dan Luo教授领导的研究人员将开发出有史以来第一个不使用活细胞的纳米技术蛋白质制造过程。研究人员将使用纳米粘土晶体沿着DNA作为起始材料;纳米粘土是一种廉价的材料，可以很容易地按比例放大，而DNA为所需的蛋白质提供了蓝图。纳米粘土和DNA将被加工成数十亿个微小的甜甜圈，这样蛋白质就可以通过这些甜甜圈有效地、连续地制造出来，所有这些都没有活的生物体，而且是大规模的。如果成功，这项研究将成为一个全新的平台，在这个平台上，蛋白质，包括那些不能由活生物体产生的蛋白质，可以相对廉价地大规模生产。该研究项目的目标是解决无细胞蛋白质制造的主要问题，这被认为是一个关键的制造挑战。研究人员已经在以下方面取得了令人兴奋的成果：1）DNA水凝胶在没有活细胞的情况下生产蛋白质，吞吐率提高了20倍; 2）使用纳米粘土晶体保护DNA并提高蛋白质产量; 3）NanoClay-DNA（NanoCD）微型甜甜圈形成制造平台。研究人员涵盖DNA生物纳米技术，电流体动力学处理和大规模系统设计，流体动力学和界面稳定性以及蛋白质工程。工业合作伙伴TeraPore Technologies和杜邦公司涵盖了蛋白质纯化和推广以及未来商业化的问题。 这项研究如果成功，将成为一种平台技术，使大多数蛋白质的基于NanoCD的，连续的，可扩展的和无细胞的制造成为可能。该项目还将展示在扩大的制造过程中整合分层材料：从分子级DNA到纳米级粘土，再到微尺度的电喷雾微甜甜圈凝胶颗粒，再到宏观尺度的连续生物反应器。这项研究将产生重大的社会影响和效益。首先，它将纳米材料（纳米粘土）和纳米生物技术（DNA纳米技术）转化为一个大规模的制造平台，可以取代通常缓慢，昂贵和劳动密集型的传统细胞蛋白质生产。此外，这些系统将为制造更多不同或难以表达的蛋白质铺平道路。该研究项目的成功完成将成为一种强大，经济和通用的技术，将被学术界和制药/生物技术行业采用。这项研究还将通过精心设计的，可行的和综合的三管齐下的计划影响教育：（1）向高中生推广;（2）扩大本科生和研究生的经验和视野，包括国际研究经验和工业经验;（3）增加代表性不足的少数民族和女性工程专业学生的招聘。

Proteins are becoming increasingly important drugs to treat and prevent diseases. In fact, among the current top ten best selling drugs in the world, eight of them are protein-based. At present, almost all proteins are manufactured by living organisms. Unfortunately, using living organisms to produce proteins has various limitations and problems including low production rate and high set-up cost. Most severely, living organisms need to be sufficiently fed and well taken care of, which has not been easy on an industrial scale. In this project, the researchers led by Prof. Dan Luo at Cornell University will develop a first-ever, nanotechnology-enabled protein manufacturing process without using live cells. The researchers will use nanoclay crystals along with DNA as starting materials; nanoclay is an inexpensive material that can be easily scaled up, and DNA provides the blueprint for the desired proteins. Together, nanoclay and DNA will be processed into billions of teeny tiny donuts such that proteins can be manufactured through these donuts efficiently and continuously, all without living organisms and on a large scale. If successful, this research will become an entirely new platform where proteins, including those that cannot be produced by living organisms, can be manufactured relatively inexpensively on a large scale. The goal of this research project is to address major issues for cell-free protein manufacturing, which is recognized as a critical manufacturing challenge. The investigators have established exciting results in: 1) DNA hydrogels to produce proteins without live cell with 20X enhanced throughput rates; 2) use of nanoclay crystals to protect DNA and enhance protein productions; 3) NanoClay-DNA (NanoCD) microdonuts to form a manufacturing platform. The investigators covers DNA bionanotechnology, electro hydrodynamic processing and large-scale system design, fluidic dynamics and interface stability, and protein engineering. The industrial partners, TeraPore Technologies and DuPont, cover issues for protein purification and outreach and future commercialization. The proposed research, if successful, will become a platform technology that enables NanoCD-based, continuous, scalable and cell-free manufacturing of most proteins. The project will also be a demonstration of integrating hierarchical materials in a scaled-up manufacturing process: from molecular-scale DNA to nano-scale clay to micro-scale electrosprayed micro-donut gel particles and to macro-scale continuous bioreactors. This research will have significant societal impact and benefits. First, it translates both nanomaterials (nano-clay) and nanobiotechnology (DNA nanotech) into a large-scale manufacturing platform that may replace the often slow, costly, and labor-intensive conventional cell-based protein productions. In addition, these systems will pave the way for manufacturing many more different or difficult-to-express proteins that will be urgently needed. The successful completion of this research project will have become a powerful, economic and universal technology that will be adopted by both academics and pharmaceutical/biotechnology industries. This research will also impact education with a carefully-designed, feasible, and integrated three-pronged plan: (1) Outreach to high school students; (2) Expand undergraduate and graduate students' experience and horizons including international research experience and industrial experience; and (3) Increase the recruitment of under-represented minority and female engineering students.