Practical Applications of Biofluorescent Particle Counting in Environmental Monitoring Investigations

Introduction to Biofluorescent Particle Counting

Biofluorescence particle counting (BFPC) is a relatively new rapid microbiological method for monitoring airborne microbes (1, 2), providing continuous information on particle count, size, and autofluorescence (3). The technology is based on laser excitation of particles to detect biofluorescence of biological compounds, such as nicotinamide adenine dinucleotide, riboflavin, and picolinic acid, which are universally found in all microorganisms. Currently, some BFPC systems cannot distinguish between viable particles and other compounds (such as polymers or solvents) that may fluoresce; to help distinguish false positives from viable cells, the use of qualitative sample capture onto media or filters can help determine microbial identification (4). The instruments are qualified using the same principles as traditional particle counters. The BFPC systems provide instant and continuous monitoring without the need for reagents or incubation. BFPC systems use the autofluorescence unit (AFU) as an alternative to the traditional colony-forming unit (CFU). An exact agreement between AFU and CFU counts should not be expected, as the detection methods are different. Because the AFU is based on cellular detection and the CFU is dependent on observance of microbial growth, AFU counts are generally higher than CFU counts. Moreover, it is estimated that only 0.1–1.0% of microbial cells are culturable (5). AFU results that are culturable and viable but not culturable cells are expected to be much higher than the traditional CFU results. Therefore, AFU counts that are greater than CFU counts should not lead to the automatic conclusion that the environment is out of control, nor does it imply that there is more risk for contamination. Correlating CFUs to AFUs is difficult, as most microorganisms in the environment are not culturable and, therefore, undetectable with traditional plate-count methods. Traditional plate-count methods are well-established strategies used for decades to demonstrate microbial control of pharmaceutical manufacturing facilities. It is, however, useful to make side-by-side empirical comparisons because these data can provide a deeper understanding of the technology's performance in manufacturing operations and will help establish control and which will be required for establishing the BFPC method for use in routine environmental monitoring (EM) (6, 7).

The authors believe that BFPCs can be used in a number of scenarios in aseptic pharmaceutical manufacturing, including for continuous microbiological EM to increase process control and reduce interventions and contamination control strategies. These applications are outlined in the Other Applications section later in this article. This article focuses on investigations.

Opportunities for the Use of BFPC for Investigations

This article has been written by 20 subject matter experts from 17 companies, facilitated by the BioPhorum Operations Group Alternative and Rapid Microbiological Methods Collaboration. Although not having a traditional analog, BFPC technology provides real-time direct detection of viable microorganisms in air samples. It gives immediate results with a time stamp that can lead to a quicker identification of the root cause of excursions, ultimately allowing for quicker product manufacturing decisions, instead of waiting three to five days for traditional microbial results. Therefore, BFPC is a useful investigational tool. This article provides guidance on using the technology in this way. A case study is presented, in which BFPC is used to identify the source of a mold excursion in a short timeframe.

BFPCs enable a more streamlined investigative process, as root cause investigations into EM excursions are traditionally based on results from growth-based methods, requiring time-consuming incubation. As a result, investigations cannot start for several days after the actual event occurred. By then, the process may have been completed and the affected areas cleaned. The lack of immediacy in traditional approaches also prompts an overreliance on the recollection of personnel about possible causes of excursions in busy operational environments; sources of EM excursions can also be inconclusive. Unidentified sources of contamination often result in corrective and preventative actions (CAPAs), including increased sampling, cleaning, and training; however, it is not always clear if the true root cause has been found. Because BFPC systems do not require incubation, rapid characterization of the manufacturing space is possible. The instrument can be moved around the room to immediately detect AFUs with timestamps and to run continuously for detection of transient events, thereby enabling significantly faster investigations. BFPC provides insights leading to more appropriate CAPAs, which can be subsequently verified with traditional microbiological methods for culturable organisms. Of course, traditional methods may verify the presence of organisms only if the organisms initially detected by the BFPC (which leads to a CAPA) can be recovered and cultured.

Proposed Method for Investigations Using BFPC

Several vendors provide commercial BFPC systems. The units typically provide viable particle counts, total particle counts, and integrated particle collection (sample capture) functionality into a single portable instrument. As outlined above, these systems have the potential to be used for routine EM, but to be clear, the application described in this article is for using BFPC as a supplement to routine regulatory stipulated EM, enabling risk reduction and speedier right-first-time investigations, not as a replacement for routine monitoring. In an investigation, BFPC can be used in two modes to identify the source of an excursion: mapping the overall area followed by scanning particular suspect areas, and trending.

We recommend the following approach.

Case Study: Identification of a Contamination Source

The following example demonstrates how one BioPhorum member company used the technology to support a contamination investigation. They were investigating recovery of mold that appeared on active air sampler plates used for routine environmental air sampling in a grade D cleanroom. The suspected sources were addressed by corrective actions before this specific investigation; however, mold was subsequently detected. The company decided to use BFPC to continue the investigation.

To provide baseline information, the BFPC unit was placed in the center of the area of interest and run overnight for 902 min. The average overnight recorded viable count was 9.01 AFUs/m³. The next day, the area was mapped by sampling for 35 min (1 m³ of air) in each of five locations. The results were 15, 31, 32, 38, and 54 viable counts/m³, respectively. The investigations team focused on the location with the highest count. HEPA filters were located in the ceiling above the location, and the team suspected that they were the source of the contamination. The BFPC unit was fitted with the “sniffer” tube, and all the HEPA filters in the room were scanned. No leaks of viable particles from the filters were detected. The device was then used to rapidly scan the entire area near the location. This quickly identified an eye wash shower station as the likely source of the viable particles. A further sample of 1 m³ of air was then acquired at the station, directing the inlet toward the airflow adjacent to the shower handle, giving a count of 95 viable counts/m³ (see Figure 1). A gelatin filter to collect the detected microorganisms was also used, and a mold isolate was subsequently identified in the sample. This example demonstrates the effectiveness of the BFPC unit for the investigation team to quickly identify—in real time—that the eye wash station was the probable source of the excursion. As a corrective action, all eye wash stations in the area were remediated to prevent interstitial air from entering the containment suite. The BFPC unit was later used to confirm that the remediation on the eye wash shower stations had been effective.

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Figure 1

Eye wash shower handle. Particle counter minute-by-minute continuous sampling at a flow rate of 0.028 m³/min. Total particulate counts are on the right scale, and viable counts on the left scale. Total viable count of 95.

Business Benefits of Using BFPC for Investigations

BFPC provides clear and rapid results to allow effective decision-making and efficient responses. In the root cause investigation case study, the BFPC unit was used to identify the source of the mold excursion on the second day of the investigation. Using a BFPC allowed the investigation to be completed significantly quicker than traditional procedures (60 days, typically), reduced the time that the manufacturing suite sat idle, and increased the capacity of manufacturing days. Another site achieved a hard saving of $176,000 annually by reducing superfluous and hazardous sporicidal treatments. In another case, a BioPhorum member company completed an investigation five days earlier than it would have been with traditional methods, enabling the site to manufacture two engineering batches for an upcoming product launch.

The cost of a BFPC unit is in the region of $50,000–$85,000, and the authors think that access to a BFPC unit has the clear potential to provide a significant return on investment and reduce the risk to product quality. Although the economic value of a “day saved” varies considerably depending on the operational and supply chain context, biopharmaceutical manufacturing facilities are relatively costly to operate. As an indicator, consider that the company of one of the authors estimates that an aseptic filing suite costs about $300,000 per day to run. Therefore, closing an investigation early by even a few days and preventing further excursions by getting to the root cause the first time will generate substantial economic benefits exceeding the cost of the BFPC unit.

Other Applications of BFPC