Rapid Microbiological Methods (RMM) have intrigued pharmaceutical manufacturers for several years. The benefits of RMM are well established; they enable better insight into the manufacturing process by providing microbial information much faster than the compendial methods that take multiple days to provide results. Most currently available RMM instruments are laboratory-based and significantly reduce the time to obtain microbial results from a collected sample. Several manufacturers have recently introduced real-time viable particle detectors similar to the BioTrak Real-Time Viable Particle Detector shown in Figure 1.
Real-time viable particle detectors use optical techniques to determine particle viability on a particle-by-particle basis. This capability provides for:
- trending of microbial content in the manufacturing environment;
- instantaneous notification of microbial contamination events;
- enabling segregation of potentially exposed product
- rapid initiation of root cause investigations
- providing input to Process Analytical Technology (PAT) based process control measures.
- potential for real-time product release
While real-time viable particle counters offer significant potential benefits, they also present some new challenges to industry and regulators. This article will address the unique challenges associated with evaluating, testing, and validating this new family of RMM instruments.
|Figure 1. TSI BioTrak Real-Time
Viable Particle Detector
Real-Time Viable Particle Detector: Principles of Operation
Real-time viable particle detectors use the intrinsic fluorescence of microbial constituents associated with cell viability to analyze environmental particles entrained in the aerosol sample flow path. When excited by ultra violet laser light, the metabolites associated with cell viability fluoresce and emit light at a higher wavelength than the excitation wavelength. Principal microbial fluorophores associated with cell viability are tryptophan (excitation peak ~280 nm, emission peak ~340 nm), NADH (excitation peak ~340 nm, emission peak ~450 nm), and riboflavin (broad excitation ~350-450 nm, emission peak ~530 nm).
The most common excitation source for portable viability detectors is the 405 nm laser diode. The technique is non-specific. Organism specificity is not obtainable due to the complex composition of the viability metabolites and the resulting fluorescence signal. Laser induced fluorescence (LIF) has been used in military and homeland defense threat detection products since the late 1990’s. It has only recently been adapted for use in the pharmaceutical manufacturing environment.
Real-time viable particle detectors have several key operating parameters:
- Sample Flow Rate
- Aerosol Efficiency
- False Positive Rate
Sample flow rate is the amount of air that the instrument analyzes. Higher flow rates enable better characterization of manufacturing environments and reduce the time required to meet mandated sample volume requirements. Aerosol efficiency is the ratio of the number of particles present in the manufacturing environment compared to the particles that reach the instruments analysis engine. Sensitivity and False Positive Rate describe the ability of the real-time viable particle detectors to measure and differentiate viable particles from non-viable particles. Sensitivity is the ability to measure low numbers of viable particle counts. A false positive occurs when a non-viable particle is classified as a viable particle. False positives can occur due to the non-specific nature of the LIF technique. Additionally, non-viable particles such as pollens, skin flakes, and paper dust have fluorescence properties and create optical signals that must be addressed during instrument design. Typically, higher sensitivities result in higher levels of false positives. These key operating parameters should be considered and addressed when evaluating and validating real-time viable particle detectors.
Due to the low intensity of the fluorescence signals, real-time viable particle detectors that have sample flows greater than approximately 5 liters/minute must reduce the flow to increase the fluorescence emission intensity. Aerosol concentration is the enabling technology incorporated into high flow rate detectors. Aerosol concentrators utilize particle inertia to concentrate the larger particles of interest into a lower velocity flow volume. The incoming sample flow is separated into a high (major) volume flow that is exhausted from the system and a low (minor) volume flow that is analyzed. Small low-inertia particles follow the high-volume flow path while larger high-inertia particles follow the low-volume analyzed flow path. Figure 2 illustrates the operating principles of a concentrator.
Figure 2. Aerosol Concentration: Small particles follow the major flow path and are not analyzed; large particles with greater inertia follow the minor flow and are subsequently analyzed.
Two key parameters define concentrator performance: aerosol efficiency and D50 cut point. Figure 3 shows aerosol efficiency on the Y-axis and particle size on the X-axis. Aerosol efficiency is the ratio of the particles in the minor (analyzed flow) flow versus the total number of particles present at the inlet of the concentrator at each given size.