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越来越重要的药物。事实上，在目前世界上最畅销的10种药物中，有8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，使用生物体生产蛋白质有各种限制和问题，包括低生产率和高设置成本。最严重的是，生物需要得到充足的食物和良好的照顾，这在工业规模上并不容易。在这个项目中，由康奈尔大学的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工艺。研究人员将使用纳米粘土晶体和DNA作为起始材料；纳米粘土是一种廉价的材料，可以很容易地放大，DNA提供了所需蛋白质的蓝图。纳米粘土和DNA将一起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高效、连续地制造出来，而不需要生物体，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。研究人员在以下方面取得了令人兴奋的成果：1)DNA水凝胶可以在没有活细胞的情况下产生蛋白质，其吞出率提高了20倍；2)利用纳米粘土晶体保护DNA，提高蛋白质产量；3) NanoClay-DNA （NanoCD）微甜甜圈形成制造平台。研究方向包括DNA生物纳米技术、电流体动力学处理与大规模系统设计、流体动力学与界面稳定性、蛋白质工程等。工业合作伙伴TeraPore Technologies和杜邦（DuPont）负责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，将成为一个平台技术，使基于纳米ocd的、连续的、可扩展的和无细胞的大多数蛋白质制造成为可能。该项目还将展示在大规模制造过程中整合分层材料：从分子尺度的DNA到纳米尺度的粘土，再到微观尺度的静电喷涂微甜甜圈凝胶颗粒，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迫切需要的不同或难以表达的蛋白质铺平道路。这项研究项目的成功完成将成为一项强大、经济和通用的技术，将被学术界和制药/生物技术行业采用。本研究还将以一个精心设计的、可行的、综合的三管齐下的计划来影响教育：(1)扩展到高中学生；(2)拓展本科生和研究生的经验和视野，包括国际研究经验和行业经验；(3)增加招收代表性不足的少数民族和女性工程专业学生。

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.