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Pharmaceutical Manufacturing

Q: What do BioVigilant’s Instantaneous Microbial Detection systems sample?

A: The IMD-A samples air and the IMD-W samples water.

Q: What types of microbes can BioVigilant’s IMD-A systems detect?

A: Bacteria, yeast, and mold.

Q: Do BioVigilant’s IMD systems count spores as microbes?

A: Yes. Both systems will detect spores and count them as microbes.

Q: How do IMD systems detect microbes?

A: BioVigilant’s IMD systems detect the presence of microbes by simultaneously measuring the size and intrinsic fluorescence of every particle in an aerosol/liquid sample as it is drawn through the instrument and interrogated by a laser. Microbes possess certain organic auto-fluorescent compounds necessary for cellular function, including NADH, riboflavin, and DPA (in the case of endospores). When these intrinsic fluorophores are excited by laser, they auto-fluoresce and this signal is measured. The IMD systems use this fluorescence measurement as a critical metric within the proprietary algorithms to differentiate a particle of biological origin (e.g. a bacterium) from an inert (e.g. dust) particle.

Q: What specific applications are ideal uses for an IMD-A or IMD-W system?

A: IMD-A systems are ideally suited wherever timely information about a critical environment is important. IMD-A systems set a standard for speed in rapid microbial detection, delivering continuous real-time data about one’s environment empowering the user to detect, react to, and prevent contamination with a sensitivity not previously achievable with traditional methods. The most common IMD-A applications involve the monitoring of critical environments (i.e. cleanrooms) in the Pharmaceutical manufacturing and other Life-Science industries. Aseptic fill lines, sterility test suites/isolators, and RABS areas are all key locations. Other specialized applications include personnel training, compressed gas testing, process simulations (e.g. water and media fills), root-cause investigations (e.g. sourcing of building mold contamination), engineering validation (e.g. determining optimum HVAC settings) and risk assessment/process improvement (the synchronized IMD-A webcam works great for this application).

Similarly, IMD-W instruments deliver value through instantaneous and continuous results about bioburden in purified water. There are multiple application scenarios within a typical Purified Water or Water for Injection production facility, including pretreatment and RO effectiveness testing, storage tank sampling, point-of-use sampling, and other routine water sampling points. Many of these scenarios deliver value by providing a continuous risk reduction mechanism, but other benefits can include energy savings from reduced sanitization cycles, improved resource allocation by reduced sampling frequencies, and improved process understanding.

BioVigilant’s team of Field Application Scientists is dedicated to helping customers achieve implementation success in these general and more specialized applications. Please contact us to explore your requirements.

Q: In what ways are BioVigilant’s instruments preferred to conventional plate culturing methods and other “rapid microbiological methods”(RMMs)?

A: The primary ways in which BioVigilant’s IMD-A and IMD-W systems are preferred to conventional plate culturing methods and other rapid methods are: 1) time from measurement to results is virtually zero; 2) monitoring can be continuous as opposed to episodic; 3) in most cases, there is minimal or no time required to set up the sample; and, 4) there is minimal human intervention.

Q: What testing has been done to assure IMD-A and IMD-W systems are reliable instruments?

A: As BioVigilant’s flagship product, IMD-A systems underwent rigorous testing against the USP Validation of Alternative Microbiological Methods guideline conducted in one of the world’s most advanced aerosol test chambers, custom built for bacterial challenge testing. The test results and protocol were reviewed with the FDA and have been submitted with BioVigilant’s Drug Master Files. For a summary of these results, please ask your salesperson. In addition, the systems are certified by TUV Rheinland for compliance to all relevant standards.

As a newer product, the IMD-W system has undergone an initial battery of tests in accordance with recommendations set forth by the Online Water Bioburden Analyzer (OWBA) industry work group. Further testing is planned based upon end user requirements for their specific application. The IMD-W system is also certified by TUV for compliance to relevant standards.

Q: What do the regulatory agencies say about implementing IMD-A and IMD-W systems in pharmaceutical applications?

A: Regulatory agencies are strong proponents of RMMs, as evidenced by their Process Analytical Technologies (PAT) Initiative, and have expressed the desire to see RMMs more widely used by pharmaceutical companies. Regulators understand and accept the possibility that these newer, more sensitive technologies may yield a different quantity and quality of data, including detection of higher counts than previously observed with traditional methods. This higher sensitivity provided by the new methods is actually advocated because it allows for a greater understanding and control of the environment. They have also indicated that previously established limits (e.g., for environmental monitoring) may need to be adjusted due to the possible disparity/increase in counts of the more sensitive RMM from that of the traditional method. The FDA and EMA regulators are very willing to work with users on validation and implementation plans. BioVigilant maintains open communication with both regulatory bodies including educating inspectors and regulators of both bodies, and is happy to share its experiences with customers to facilitate communication.

Q: Many instruments detect the size of particles, some detect fluorescence, and some even do both sequentially. Why is it necessary that size and fluorescence be measured simultaneously, and for each particle individually?

A: If measurements of particle characteristics come from different instruments, or even at different times from the same instrument, it is very difficult to correlate the measurements in such a way that advanced deterministic processing algorithms can be applied. Such algorithms and correlated measures achieve maximum sensitivity while simultaneously minimizing any possible influence of electronic “noise” and are crucial, for example, to determine whether detected particles are inert or microbiological in nature. IMD systems use proprietary algorithms to analyze the size and fluorescence information from each particle in real time to provide deterministic results.

Q: What is fluorescence and why is fluorescence relevant to microbial detection?

A: Fluorescence is the emission of light at longer wavelengths by a substance after it absorbs incident light at shorter wavelengths. Microbes such as bacteria and fungi consist of complex organic chemical compounds such as proteins and amino acids necessary for cellular metabolism, which are capable of fluorescence emission under the excitation of a suitable light source such as laser light illumination. BioVigilant’s IMD systems detect microbes by measuring this type of fluorescence signal.

Q: What reagents, dyes, and growing medium are required in order to use BioVigilant’s IMD systems?

A: None. Intrinsic fluorescence is at the core of IMD technology, which enables detection without any costly reagents or labor-intensive sample preparation.

Q: What human intervention is needed to operate an IMD-A or IMD-W system?

A: The system needs to be turned on and the sample initiated through the instrument’s user interface. The instrument can be operated either continuously or episodically. In continuous mode, the sample will continue until an operator selects “stop” in the software. In episodic mode, the sample will stop automatically when the specified sample volume has been collected or the duration of time has transpired.

Q: What are the advantages of continuous monitoring as opposed to episodic monitoring?

A: (1) Continuous monitoring and real-time detection of microbes enables clean room and/or facility management to take corrective measures in a timely manner if a microbial excursion occurs; (2) continuous monitoring can provide trending data useful in isolating the root cause of a contamination problem; and, (3) continuous and real-time monitoring facilitates the implementation of FDA’s PAT initiative (Process Analytical Technology).

Q: Is it necessary to have an idea of the target microbes before deploying an IMD-A or IMD-W system?

A: No. IMD systems are often used to gain exactly such an understanding of one’s environment, and thus, gain the ability to measure and control it.

Q: Is it necessary to have an idea of the target microbes before deploying an IMD-A system?

A: No.  IMD-A systems are often used to gain exactly such an understanding of one’s environment, and thus, gain the ability to measure and control it.

Q: How quickly can an IMD system detect the presence of microbes?

A: BioVigilant’s IMD systems detect the presence of microbes instantaneously as the environmental air (IMD-A) or water (IMD-W) passes through the device. The determination of the presence of microbes is done on an individual particle-by-particle basis at a time period equal to the time it takes for a particle to pass through the laser beam– about one millisecond. The reporting of this information to the user is updated every second through the instrument’s user interface. For the IMD-A, the length of time it takes to sample a particular volume of air is determined by the flow rate, either ~0.05 CFM (1.15 LPM) or 1 CFM (28.3 LPM). For the IMD-W, the measured flow rate is 5 mL/min.

Q: What is a fluorophore?

A: In the context of fluorescence spectroscopy and biochemistry, the term “fluorophore” is often used. A fluorophore is any molecule which is capable of fluorescence emission. In the field of microbial detection, some of the relevant fluorophores in a microbe are a reduced form of nicotinamide adenine dinucleotides (NADH), and riboflavin. These fluorophores will emit fluorescence under laser light illumination and are used by BioVigilant’s IMD systems to enable microbial detection.

Q: Are BioVigilant’s IMD systems different from fluorescent-antibody and other related techniques?

A: Yes. BioVigilant’s IMD systems rely on intrinsic fluorescence (also called “auto-fluorescence”) emanating from fluorophores inside microbial cells, so no sample preparation is needed to detect microbes. This is in contrast with fluorescent dye-based techniques, whereby, in order for the microbes to be detected, the sample needs to be treated with a reagent to give out a specific fluorescence signal. This process is embodied in several common techniques, such as ATP Bioluminescence detection and flow cytometric detection.

Q: What is the chance that two or more separate microbes will pass through an IMD system’s laser so closely together that they may inaccurately be counted as one larger particulate?

A: While such a condition is theoretically possible, in operation, it is highly unlikely in any cleanroom environment (IMD-A) or purified water (IMD-W). Because particles tend to be randomly distributed and they pass through the laser interrogation zone of an IMD instrument on the order of milliseconds, the chance of two separate microbes being interrogated at the same time in a relatively clean sample is statistically low. This probability can be calculated, and when the particle concentration is high enough (greater than ~100,000 particles per liter of air for the IMD-A or greater than ~200,000 particles per mL for the IMD-W) the probability increases to the point of statistical significance. In this case, the detector may give under-sampled results. Such an effect is termed “coincidence error”, and is a common characteristic of particle measuring instrumentation. However, in properly operating cleanrooms or water systems, particle concentrations are well below this level.

Q: Can BioVigilant’s IMD systems tell the difference between live and freshly killed microbes?

A: The answer to this question depends on how much intrinsic fluorescence capacity remains in the killed microbe. If significant quantities of fluorophores are intact, the dead cell may continue to fluoresce sufficiently to be categorized as a microbe. If, however, the microbe has been dead for a while or was killed by a chemical technique which destroys the fluorophores within the cell, the amount of fluorescence remaining will be below the threshold required for the IMD system to count the particulate as a microbe.

Q: I’ve heard that RMMs, including IMD systems, exhibit greater sensitivity than traditional methods. What do I do if I get consistently higher biologic counts with the IMD system than with the traditional method?

A: There are several reasons why an IMD system may give biologic counts higher than CFU counts traditionally seen with growth-based methods. These reasons include the IMD system’s ability to monitor continuously, its greater detection sensitivity, and its ability to detect microbes that don’t grow well in the standard growth conditions and media used. Pharmaceutical industry thought leaders as well as FDA regulators agree that using RMMs may indeed yield consistently higher counts than traditional approaches. The general consensus is that comparability testing should be performed between old and new methods during the validation process, and that a robust dataset with statistical analysis can provide the key metrics necessary to implement a sound monitoring strategy. BioVigilant can assist customers with developing and executing such testing architectures.

Q: Should I be reacting to low/non-zero biologic counts on an IMD system when traditional methods historically have yielded zero CFU?

A: Many customers have found that using a more sensitive RMM such as an IMD-A or IMD-W can potentially give higher counts than historical data using growth-based methods in applications where some microbes are expected (e.g. ISO 7 rooms for the IMD-A or purified water loops for the IMD-W). However, in the most strictly-controlled environments (such as ISO 5 aseptic areas with the IMD-A or a WFI point of use with the IMD-W), the IMD system will generally corroborate traditional methods in confirming the area is well controlled and below microbial contamination limits (often resulting in zero counts with both methods). The question of whether or not to “react” depends on the IMD application, guidance derived from robust comparability testing during validation (if applicable), and the subsequent Alert and Action levels that are set. Similarly, some customers view the ability to obtain continuous, real-time data as an opportunity to create frequency-based limits instead of the traditional magnitude-based limits as is suggested in USP for environmental monitoring (e.g. ≤1% contamination recovery rate in an ISO 5 environment). In either scenario, real-time feedback empowers manufacturing personnel to understand, react, and ultimately improve control of their critical environments.

Q: Can IMD systems identify organisms? If not, what strategy should I use for deploying identification?

A: The IMD technology is unable to identify microorganisms because the intrinsic fluorophores used for detection are widely found among microorganisms, thus precluding specificity to a particular genus and/or species. Certain customers and monitoring applications have traditionally used a microbial identification approach (e.g. RT-PCR, gas chromatography, etc.) in response to a microbial excursion. This approach can still be used in tandem with IMD technology; an air sample for identification can be collected immediately upon an IMD-A detection alarm, and a water sample can be automatically collected by the IMD-W upon a detection alarm. As with any identification strategy, this is necessarily supplemental retrospective information. Timely detection and response are the paramount factors (i.e. you can’t identify microbes and perform root-cause analysis for a microbial excursion you never detected). Alternately, some customers consider microbe identification to provide less overall value to maintaining environmental control compared to using real-time data to control and improve processes.

Q: Does BioVigilant provide support for the validation process?

A: Absolutely. BioVigilant has a dedicated team of Field Application Scientists whose role is to assist customers across all stages of the evaluation, validation and implementation processes. BioVigilant can provide documentation templates (e.g. IQ/OQ documents, data analysis spreadsheet(s)), IQ/OQ services, specialized training, help conduct evaluation protocols, facilitate unique applications, assist with data analyses and generally consult on whatever the challenges may be. Furthermore, BioVigilant maintains open communication with regulatory bodies including the FDA to help foster robust dialogue and informed decision making for all parties involved in the validation process.

Q: Can the system be networked?

A: Each IMD system contains an embedded PC and I/O communication ports including Ethernet 10/100 Base-T. The user can connect to the IMD-A system directly using one of several local or remote schemes. To interface the IMD software with existing software such as SCADA or LIMS systems, custom middleware can be developed to meet the exact needs of each customer. Contact us for more information.

Q: Can the IMD-A or IMD-W system be used to monitor particles?

A: Although the primary application for IMD systems is to detect biological particles, some customers find additional value in monitoring the total particles detected. Due to their size, cost, and technological complexity, the IMD systems may not be ideal for direct replacement of aerosol or liquid particle counters in some traditional applications like episodic cleanroom certification. However, the IMD-A systems have been tested extensively against industry-leading particle counters and fare favorably. In addition to meeting certain standardized metrics for particle counters (e.g. from ISO 21501-4), the IMD-A systems commonly demonstrate exceptional correlation (R2 ≥ 0.95) with aerosol particle counters. IMD-W systems will be evaluated against liquid particle counters soon. Contact us for more information and to discuss your particular application and requirements.