A Therapeutic Cell Distillery: Light-Controlled Fractionation of Stem Cells for Next-Generation Biomanufacturing Processes

治疗性细胞酿酒厂：用于下一代生物制造工艺的干细胞光控分离

• 批准号: 1743404
• 负责人: Stefano Menegatti
• 金额: $ 38.07万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1743404&HistoricalAwards=false
• 关键词: Therapeutic; Cell; Distillery; Light; Controlled

PI: Menegatti, StefanoProposal: 1743404A major challenge in developing robust manufacturing processes for therapeutic cell products (TCPs) is being assured that the cells produced have the desired therapeutic potency. Many of the current separation and enrichment processes are based on sorting technologies using "affinity tags," which are adsorbents that bind cells specifically by targeting their surface markers. The process of removing these tags after sorting is completed is often associated with low yield and cell damage. The research goal of this project is to develop an innovative "affinity tag" based purification technology for separating complex mixtures of cells into cell fractions with different bioactivity and unique therapeutic value using light at controlled intensity and exposure time to control the binding and sequential release of different cell populations. The use of the "affinity tags" ensures that the recovered cells are highly pure, while the use of light as a biologically gentle mean to control cell binding ensures that the recovered cells are bioactive. The cell products targeted include stem and progenitor blood cells, which give rise to all the other blood cells, and can be administered by intravenous infusion into patients whose bone marrow or immune system is damaged or defective. The proposed technology is flexible, and can be easily adapted towards both research and processing scenarios, from use in basic cell biology and tissue engineering research, all the way to the production of clinically relevant amounts of cells. The interdisciplinary team encompassing the departments of Chemical Engineering, Biomedical Engineering, and the Biomanufacturing Training and Education Center (BTEC) at North Carolina State University will integrate research with a plan for workforce development and engagement with biotech companies. Activities include BTEC training opportunities for students and industrial and federal (FDA and BARDA) employees on cGMP processing of protein and cell therapeutics, incorporation of outcomes into existing BTEC courses on bioseparations, and creation of ad hoc half-semester and short courses targeted to biotech professionals on fractionation and purification of cell-based products. The project also plans to connect with stakeholders of NIIMBL, a NIST institute designed to innovate biomanufacturing in the United States, to develop joint research projects aimed at the large-scale translation of the project's cell distillery for TCP production. The project proposes to develop a technology based on light-controlled affinity to separate stem cell populations into highly pure and bioactive fractions with distinct therapeutic value. This "cell distillery" employs marker-specific peptide ligands with light-controlled binding activity integrated in a microfluidic platform, to control cell binding and release based on the expression and density of cell surface markers. The ligands are designed to target cell markers selectively, and switch reversibly between ON and OFF binding modes upon exposure to visible/near IR light at specific wavelengths. The optimization of ligand density on the devices and light dosage ensures the recovery of highly pure and viable cell fractions. The project will demonstrate the technology by (1) separating hematopoietic stem cells (HSCs) from multipotent hematopoietic progenitor cells (HPCs), followed by the fractionation of (2) HSCs into long-term and short-term HSCs, and (3) HPCs into multipotent and lineage-committed HPCs. To this end, the project will develop light-responsive ligands for VCAM-1, CD38, and Flt-3 markers (Objective 1); correlate cell binding to ligand density and light irradiance (Objective 2); construct and characterize a microfluidic "cell distillery" for fractionating HSCs and HPCs obtained from commercial whole bone marrow aspirate or cord blood mononuclear cell fractions (Objective 3). The outcomes of this research have the potential to transform the science and technology of manipulation and isolation of therapeutic cell products (TCPs), opening new avenues in regenerative medicine and treatment of challenging malignancies.

为治疗性细胞产品（tcp）开发稳健生产工艺的主要挑战是确保生产的细胞具有所需的治疗效力。目前的许多分离和富集过程都是基于使用“亲和标签”的分选技术，这是一种通过靶向细胞表面标记物特异性结合细胞的吸附剂。在分选完成后去除这些标签的过程通常与低产量和细胞损伤有关。该项目的研究目标是开发一种创新的基于“亲和标签”的纯化技术，将复杂的细胞混合物分离成具有不同生物活性和独特治疗价值的细胞组分，利用可控强度和曝光时间的光来控制不同细胞群的结合和顺序释放。使用“亲和标签”确保回收的细胞是高度纯净的，而使用光作为一种生物温和的手段来控制细胞结合，确保回收的细胞具有生物活性。目标细胞产品包括干细胞和祖血细胞，它们产生所有其他血细胞，可以通过静脉输注给骨髓或免疫系统受损或有缺陷的患者。所提出的技术是灵活的，可以很容易地适应研究和处理场景，从基础细胞生物学和组织工程研究的使用，一直到临床相关数量的细胞生产。这个跨学科团队由北卡罗莱纳州立大学化学工程系、生物医学工程系和生物制造培训与教育中心（BTEC）组成，将把研究与劳动力发展计划和与生物技术公司的合作结合起来。活动包括为学生、工业和联邦（FDA和BARDA）雇员提供关于蛋白质和细胞治疗的cGMP处理的BTEC培训机会，将结果纳入现有的BTEC生物分离课程，以及针对生物技术专业人员创建关于细胞基产品分离和纯化的临时半学期和短期课程。该项目还计划与NIIMBL的利益相关者建立联系，NIIMBL是一个旨在创新美国生物制造的NIST研究所，旨在开发联合研究项目，旨在将该项目的细胞蒸馏厂大规模翻译为TCP生产。该项目建议开发一种基于光控亲和力的技术，将干细胞群体分离成具有独特治疗价值的高纯度和生物活性组分。这种“细胞蒸馏器”将光控结合活性的标记特异性肽配体集成在微流控平台上，根据细胞表面标记物的表达和密度来控制细胞的结合和释放。该配体被设计为选择性靶向细胞标记物，并在暴露于特定波长的可见光/近红外光时可逆地在ON和OFF结合模式之间切换。设备上配体密度和光照剂量的优化确保了高纯度和活细胞组分的回收。该项目将通过(1)将造血干细胞（hsc）从多能造血祖细胞（HPCs）中分离出来，然后将(2)将造血祖细胞分离成长期造血祖细胞和短期造血祖细胞，以及(3)将造血祖细胞分离成多能造血祖细胞和谱系造血祖细胞来展示这项技术。为此，该项目将开发VCAM-1、CD38和Flt-3标记物的光响应配体（目标1）；将细胞结合与配体密度和光辐照度相关联（目的2）；构建微流体“细胞蒸馏器”，用于分离从商业全骨髓抽吸液或脐带血单个核细胞中获得的造血干细胞和造血干细胞（目的3）。这项研究的结果有可能改变治疗性细胞产品（tcp）的操作和分离的科学和技术，为再生医学和治疗具有挑战性的恶性肿瘤开辟新的途径。

项目成果

Photoinduced reconfiguration to control the protein-binding affinity of azobenzene-cyclized peptides.

• DOI: 10.1039/d0tb01189d
• 发表时间: 2020-08-26
• 期刊: Journal of materials chemistry. B
• 作者: Day K;Schneible JD;Young AT;Pozdin VA;Van Den Driessche G;Gaffney LA;Prodromou R;Freytes DO;Fourches D;Daniele M;Menegatti S
• 通讯作者: Menegatti S

Engineering Next Generation Cyclized Peptide Ligands for Light‐Controlled Capture and Release of Therapeutic Proteins

• DOI: 10.1002/adfm.202101410
• 发表时间: 2021-05
• 期刊: Advanced Functional Materials
• 影响因子: 19
• 作者: R. Prodromou;Kevin N. Day;Sahand Saberi-Bosari;John D Schneible;Matthew D. Mabe;A. San Miguel;M. Daniele;V. Pozdin;S. Menegatti

Towards continuous mAb purification: Clearance of host cell proteins from CHO cell culture harvests via “flow‐through affinity chromatography” using peptide‐based adsorbents

实现连续 mAb 纯化：使用基于肽的吸附剂，通过“流动”和“亲和层析”从 CHO 细胞培养物收获物中清除宿主细胞蛋白

• DOI: 10.1002/bit.28096
• 发表时间: 2022
• 期刊: Biotechnology and Bioengineering
• 影响因子: 3.8
• 作者: Sripada, Sobhana Alekhya;Chu, Wenning;Williams, Taufika Islam;Teten, Matthew A.;Mosley, Brian J.;Carbonell, Ruben G.;Lenhoff, Abraham M.;Cramer, Steven M.;Bill, Jerome;Yigzaw, Yinges
• 通讯作者: Yigzaw, Yinges

Molecular Engineering of Cyclic Azobenzene‐Peptide Hybrid Ligands for the Purification of Human Blood Factor VIII via Photo‐Affinity Chromatography

通过光亲和色谱法纯化人血液因子 VIII 的环状偶氮苯肽杂化配体的分子工程

• DOI: 10.1002/adfm.202213881
• 发表时间: 2023
• 期刊: Advanced Functional Materials
• 影响因子: 19
• 作者: Prodromou, Raphael;Moore, Brandyn David;Chu, Wenning;Deal, Halston;San Miguel, Adriana;Brown, Ashley Carson;Daniele, Michael Angelo‐Anthony;Pozdin, Vladimir Aleksandrovich;Menegatti, Stefano
• 通讯作者: Menegatti, Stefano

Affordable Microfluidic Bead-Sorting Platform for Automated Selection of Porous Particles Functionalized with Bioactive Compounds

经济实惠的微流珠分选平台，用于自动选择生物活性化合物功能化的多孔颗粒

• DOI: 10.1038/s41598-019-42869-5
• 发表时间: 2019
• 期刊: Scientific Reports
• 影响因子: 4.6
• 作者: Saberi-Bosari, Sahand;Omary, Mohammad;Lavoie, Ashton;Prodromou, Raphael;Day, Kevin;Menegatti, Stefano;San-Miguel, Adriana
• 通讯作者: San-Miguel, Adriana