Traditional process of sterility testing requires a 14-day sample incubation in different media. Thatmakes it impossible to establish a pre-release sterility testing for PET tracers, which have half-lives of 9 to110 minutes and must be released within 1 hour after synthesis. Moreover, even with numerous controls inplace, in the past two years two of three of the largest US PET tracer manufacturers have been issuedsterility warnings from the FDA. Trace-Ability proposes a novel approach to rapid sterility testing without incubation, with sterilityresults available within 1 hour. The solution will take advantage of microfluidic chip technology and willbe completely automated and incorporated into the only commercially available and validated automatedPET tracer QC platform. Proposed rapid sterility test involves assessing viability by probing whethercellular membranes are intact, and whether there is DNA in those membranes. This will be achieved byselective fluorescent staining of up to 250 µL of sample on a microfluidic chip. To check cell viability, wewill take advantage of two stains. The difference of the two signals will indicate whether the organism maybe able to reproduce. Proposed solution is intended to enable automated sterility testing and include it intopre-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 anddifferentiability of viable cells. A combination of stains will be selected that can differentiate between viableand non-viable organisms for 10 typical bacterial species. Evaluation criteria: (1) Viability stainsincorporate within 30 minutes, (2) Organisms retain viability after staining, (3) Stains for nonviableorganisms 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 Aim2: Development of microfluidic analysis chip for Signal to Noise (S/N) ratio optimization. Taking theoptimal stains established in aim 1, these will then be mixed with bacteria in low concentration and loadedonto microfluidic chips with varying channel sizes and shapes while monitoring fluorescence emission forbackground reduction and S/N increase. Evaluation criteria: (1) a limit of detection (LOD) of 1 CFU, (2) a100-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 implementingthe test into the Tracer-QC platform. This will allow PET manufactures to have a complete QC package ina single automated system that meets all compliance criteria. This work can extend well beyond PET tocompounding pharmacies and other small-scale manufacturers, offering simplification of the experimentalsetup, procedural changes in production, and regulatory changes to improve patient safety.
Public Health Relevance Statement: Narrative
Proposed project addresses a major risk in manufacturing of short-lived radio-pharmaceuticals for PET
- absence of a sterility test that is fast enough to be implemented before the product's rapid expiry. Solution
takes advantage of a combination of latest innovations in stain development, microfluidics and data
analysis. By enabling pre-release testing for the first time in history of PET, it this solution expected to
have an immediate benefit to public health and find applications in other fields.
Project Terms:
