Rapid pre-release sterility test for PET Drugs.

摘要

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.