Rapid pre-release sterility test for PET Drugs.

PET 药物的快速发布前无菌测试。

• 批准号: 10258453
• 负责人: Arkadij Elizarov
• 金额: $ 16.81万
• 依托单位: TRACE-ABILITY, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10258453
• 关键词: Rapid; pre; release; sterility; test

Abstract Traditional process of sterility testing requires a 14-day sample incubation in different media. That makes it impossible to establish a pre-release sterility testing for PET tracers, which have half-lives of 9 to 110 minutes and must be released within 1 hour after synthesis. Moreover, even with numerous controls in place, in the past two years two of three of the largest US PET tracer manufacturers have been issued sterility warnings from the FDA. Trace-Ability proposes a novel approach to rapid sterility testing without incubation, with sterility results available within 1 hour. The solution will take advantage of microfluidic chip technology and will be completely automated and incorporated into the only commercially available and validated automated PET tracer QC platform. Proposed rapid sterility test involves assessing viability by probing whether cellular membranes are intact, and whether there is DNA in those membranes. This will be achieved by selective fluorescent staining of up to 250 µL of sample on a microfluidic chip. To check cell viability, we will take advantage of two stains. The difference of the two signals will indicate whether the organism may be able to reproduce. Proposed solution is intended to enable automated sterility testing and include it into pre-release automated QC workflows for the 1st time in history of PET. Specific Aim 1: Selection of fluorescent stains that can provide the high selectivity and differentiability of viable cells. A combination of stains will be selected that can differentiate between viable and non-viable organisms for 10 typical bacterial species. Evaluation criteria: (1) Viability stains incorporate within 30 minutes, (2) Organisms retain viability after staining, (3) Stains for nonviable organisms have twice the binding affinity of the viability stains, being able to displace any free DNA. (4) bound:unbound ratio > 1000. (5) Limits of Detection down to 10 CFU (colony-forming unit). Specific Aim 2: Development of microfluidic analysis chip for Signal to Noise (S/N) ratio optimization. Taking the optimal stains established in aim 1, these will then be mixed with bacteria in low concentration and loaded onto microfluidic chips with varying channel sizes and shapes while monitoring fluorescence emission for background reduction and S/N increase. Evaluation criteria: (1) a limit of detection (LOD) of 1 CFU, (2) a 100-fold improvement in S/N, (3) filling times less than 30 minutes for 250 μL. Once feasibility is verified in Phase 1, this work has a clear path to commercialization by implementing the test into the Tracer-QC platform. This will allow PET manufactures to have a complete QC package in a single automated system that meets all compliance criteria. This work can extend well beyond PET to compounding pharmacies and other small-scale manufacturers, offering simplification of the experimental setup, procedural changes in production, and regulatory changes to improve patient safety.

摘要 传统的无菌检测过程需要在不同的介质中孵化14天的样品。那 使得不可能建立对PET示踪剂的释放前无菌测试，这些示踪剂的半衰期为9%到9% 110分钟，必须在合成后1小时内释放。此外，即使有许多控件， 在过去的两年里，美国三家最大的PET示踪剂制造商中有两家已经发布了 来自FDA的无菌警告。 溯源能力提出了一种快速无菌检测的新方法，无需孵化，无菌 结果将在1小时内公布。该解决方案将利用微流控芯片技术，并将 完全自动化，并整合到唯一商业上可用的和经过验证的自动化 宠物追踪器QC平台。提议的快速无菌测试包括通过探测 细胞膜是完整的，以及这些膜中是否有DNA。这将通过以下方式实现 在微流控芯片上对高达250微米的L样品进行选择性荧光染色。为了检查细胞的存活能力，我们 会利用两个污点。这两个信号的不同将表明生物体是否可以 能够繁衍后代。建议的解决方案旨在实现自动化无菌测试，并将其纳入 在PET历史上首次实现预发布的自动化QC工作流程。 具体目标1：选择能够提供高选择性和 活细胞的可分化性。将选择一种可以区分活菌的污渍组合 非活菌为10个典型菌种。评价标准：(1)活性污渍 在30分钟内加入，(2)生物在染色后保持活力，(3)不活的污渍 生物体的结合亲和力是活性污渍的两倍，能够取代任何游离的DNA。(4) 装订：未装订比率&gt；1000。(5)检出限降至10CFU(菌落形成单位)。特定目标 2：S信噪比优化微流控芯片的研制。参加了 在目标1中建立最佳菌株，然后将这些菌株与低浓度的细菌混合并加载 到具有不同通道大小和形状的微流控芯片上，同时监测 背景降低，S/N升高。评价标准：(1)检出限(LOD)为1 cfu，(2)a S/氮比提高100倍，(3)250μL灌浆时间小于30分钟。 一旦在第一阶段验证了可行性，这项工作就有了一条明确的商业化道路，通过实现 测试进入Tracer-QC平台。这将使PET制造商在 满足所有合规性标准的单个自动化系统。这项工作可以远远超越PET，扩展到 联合药房和其他小规模制造商，提供简化的实验 设置，生产中的程序变化，以及为提高患者安全而进行的法规变化。