Rapid pre-release sterility test for PET Drugs.

摘要 传统的无菌检测过程需要在不同培养基中孵育14天的样品。的 使得不可能建立PET示踪剂的预释放无菌性测试，PET示踪剂的半衰期为9至10天。 110分钟，必须在合成后1小时内释放。此外，即使有许多控制， 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行了 FDA的无菌警告 Trace-Ability提出了一种新的方法来快速无菌测试，无需孵育， 1小时内可获得结果。该解决方案将利用微流控芯片技术， 完全自动化，并纳入唯一的商用和经验证的自动化 PET示踪剂QC平台。拟议的快速无菌检查包括通过探测是否 细胞膜是否完整，以及这些膜中是否有DNA。为实现这些目标将 在微流控芯片上对多达250 µL的样品进行选择性荧光染色。为了检查细胞活力，我们 会利用两个污点这两个信号的差异将表明生物体是否可能 能够繁殖。建议的解决方案旨在实现自动无菌测试，并将其纳入 PET历史上首次推出预发布自动化QC工作流程。 具体目标1：选择能够提供高选择性和高灵敏度的荧光染料， 活细胞的分化能力。将选择一种染色剂组合， 和10种典型细菌的非活菌。评价标准：（1）活菌染色 在30分钟内掺入，（2）染色后生物体保持活力，（3）染色后无活力 生物体具有两倍于活力染色剂的结合亲和力，能够置换任何游离DNA。（四） 结合：未结合比率> 1000。(5)检测限低至10 CFU（菌落形成单位）。具体目标 2.用于信噪比（S/N）优化的微流控分析芯片的开发。以 目标1中确定的最佳菌株，然后将其与低浓度的细菌混合并加载 在具有不同通道尺寸和形状的微流控芯片上，同时监测荧光发射， 背景降低，信噪比提高。评价标准：（1）检测限（LOD）为1 CFU，（2）a 100-S/N倍数提高，（3）250 μL灌装时间小于30 min。 一旦在第1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， 将测试导入Tracer-QC平台。这将使PET制造商能够拥有完整的QC包， 满足所有合规标准的单一自动化系统。这项工作可以远远超出PET， 复合药店和其他小规模的制造商，提供简化的实验 生产中的设置、程序变更和法规变更，以提高患者安全性。

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.