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An optical technology has been developed that can determine the quantity and size of particles in liquid or air, and simultaneously determine whether each particle is inert or biologic, all in real time. The technology, developed by BioVigilant, provides instantaneous microbial detection, thereby creating wide-ranging and profound positive consequences for pharmaceutical manufacturers.

This article briefly discusses the problems this technology solves, how the technology works, and how it is applied.

How It’s Done Now and the Consequences
For both internal and regulatory reasons within the pharmaceutical manufacturing environment, it is required that testing be performed in order to determine the level of microbial contamination. Existing conventional testing methods, however, all have several undesirable attributes in common:
 

  • The cost per test is high;
  • The process is labor intensive often requiring significant set-up, monitoring, and counting;
  • The process is episodic and slow, with results generally not available for two to five days.

Among these undesirable attributes, the consequences of waiting for results are generally the most significant, and include costly planned and unplanned halts in production, as well as continued production under incorrectly assumed to be acceptable conditions and finding out later that, in fact, the conditions were not within regulatory or internal requirements. This can then result in product being thrown out, as well as make it required that expensive and time-consuminginvestigations be conducted.

The Technology
The technology was originally developed to meet the demanding specifications of the U.S. Military and Homeland Security for real time detection of the presence of airborne weaponized bio-agents such as Bacillus anthracis, which is in the size range of 1 to 7 microns. After considering the technical requirements and analyzing the suitability of existing technology for particle detection and sizing (much of which was invented 20 or more years ago), it was decided that the existing methods contained fundamental design limitations that would keep the development company from accomplishing its task. A new approachwith a fundamentally different design was required.

Using an innovative optical design, a method was invented to count and size very small particles to a sensitivity level and at lower costs not possible using existing technology. As an additional benefit, this new technology allowed the concurrent determination for each individual particle, identifying it as inert or biologic. In addition to its application within homeland security, this combination of technologies is now being applied to the field of pharmaceutical manufacturing. The following is a brief description of fluorescence sensor technology developed with regard to airborne monitoring (liquid monitoring is very similar), using an IMD-A detector. (The model IMD-A signifies “Instantaneous Microbial Detector–Air”. A similar product IMD-L was also developedwhere “L” represents liquid.)

The IMD-A consists of three components: (1) an optical assembly to measure individual particle size; (2) a concurrent optical assembly to detect a UV laser-induced fluorescence signal from certain metabolites inside microbial cells and spores; and (3) an algorithm for differentiating airborne microbes from inert dust particles.

The optical assemblies use the well-known and often used Mie scattering detection scheme [1], but apply it in a novel way, enabling the devices to make highly accurate measurements of airborne particles with size ranges from 0.5 microns to 20 microns. This capability to make fine distinctions in size is important in order to determine the class of microbe, since different classes of microbes have different size ranges, as shown in Figure 1.

The technology’s unique use of Mie scattering also facilitates the use of UV light illumination, to concurrently examine each particle for the presence of the metabolites NADH and riboflavin, which are necessary intermediates for metabolism of living organisms, and therefore exist in microbes such as bacteria and fungi. If these chemical compounds exist in a bio-aerosol, they are excited by the UV photon energy and subsequently emit auto-fluorescence light which is detected by the IMD-A. While the technology is not capable of identifying the genus or species of microbes, and viruses are too small and lack the metabolism for detection, the ability to simultaneously, and for each particle, determine the size of the particle and if it is biologic or inert, indicates to the user the presence or absence of microbial contamination.


Figure.1. Particle size ranges of several airborne inert and microbial particulates

How Microbial Detection Devices Can Be Applied to Pharmaceutical Manufacturing

The IMD-A and IMD-L devices can be used in pharmaceutical manufacturing cleanrooms as (I) warning devices; (II) as continuous monitoring and trending devices; and, (III) to verify if remediation was successful.

(I) As Warning Devices
The IMD-A can sample the air continuously or do spot checks and give an indication or alarm when microbes are detected. Figures 2 and 3 are typical device data displays. Figure 2 shows a clean air sample with no microbes, whereas Figure 3 shows the display when a burst of baker’s yeast (Saccharomyces cerevisiae)powder was sprayed in the air.

In these sample displays, airborne particle distribution histograms are shown, where the x-axis represents particle size range and the y-axis represents the particle counts per liter of air. The yellow bars in both displays denote inert particles, while the red bars in Figure 3 denote the presence, size and count of microbes. An alarm protocol can alert facility managers in the event of microbial detection, according to specific cleanroom requirements.


Figure. 2. Typical data display of clean air


Figure 3. Example display when baker’s yeast powder was present in anair sample

(II) As Continuous Monitoring and Trending Devices
The ability of the IMD devices to provide instantaneous microbial detection also enables them to provide continuous monitoring and trending, which is useful functionality not possible using existing conventional methods. In addition to helping to comply with existing regulatory and internal requirements, this unique feature of the technology makes its devices especially suitable for implementation of the FDA’s Process Analytical Technologies (PAT) initiative by providing a process analyzer tool for microbiological monitoringof cleanroom air and liquids.

In an aseptic manufacturing facility, it is advantageous to be able to observe trending patterns of microbial and particulate distribution so that a microbial incursion can be detected and action taken as soon as possible. The IMD devices are able to obtain the trending from analyses of a continuous stream of data, and thereby spot anomalies.

As an example of this application, Figure 4 shows a histogram of continuous monitoring of indoor airborne particulate matter over a 24 hour period. This time-resolved particulate distribution enables the following observations: (1) the data indicate a clear diurnal variation; (2) particle distribution is influenced by human activities as evidenced by a decrease of particle concentration after 5:30 PM and an increase at around 7:15 AM; and, (3) mechanical disturbances are also a cause of particle concentration change, as shown by a periodic change of particle count (approximately every 45 minutes) in day time, which corresponds to the periodic switching on and off of the air ventilation system.

The trending pattern revealed in Figure 4 brings to light several useful applications for pharmaceutical manufacturing: (a) any deviation from the background distribution in a controlled environment will signal a failure (or an imminent one) in the containment or filtration system; (b) strategically placed IMD-A devices can provide a two-dimensional map of microbial or particulate distribution to pinpoint the location of a contamination incursion; (c) IMD-A devices can be used as entryway or sensitive area sentries to alert for the presence of improperly sanitized personnel; and (d) IMD-A devices can be used to discover deviations from standard operating procedures by human operators.


Figure 4. Graph of environmental particle size distri-
bution histogram of an indoor office space over a
24-hour period; the particle concentration (quantity
of particles/liter of air) is represented in the gray
scale. The red lines show contour lines of equal con-
centrations of particles.

Additionally, because of the technology used by the IMD devices, samples are not destroyed. As a consequence:

• Ongoing samples may be taken, thereby making the sampling more representative of the entire batch and any contamination that exists more likely to be discovered earlier in the production process; and,

• at certain points in the production process (such as when vials are being filled), continuous sampling of entire batches is a possibility, thereby making it more likely that, if contamination is encountered, a smaller batch of production will have to be discarded than would be otherwise.

Feature Instant Microbial Detection Method Conventional Plate Culture Method Consequences
Time from measurement to results Instantaneous. Typically from one to five days Using the conventional method creates more planned and unplanned halts in production and greater potential for contamination, significantly increasing costs and lowering production. Conventional methods also are very slow to certify if remediation was successful, causing slow downs and making it more difficult to determine the cause of contamination.
Level of detection Detection and sizing of viable microbes. No identification of microbial types or viruses. Microbial detection and identification.

When using the IMD device, for those applications that require speciation, culturing would have to be done after the IMD device detected contamination.

Mode of detection Continuous monitoring and real time feedback results. Episodic monitoring and time-delayed feedback of results. Continuous monitoring increases accuracy, allows for trending to spot problems early, lowers chances of contamination, reduces the need to dispose of production, reduces down time and the time needed to remediate, and conducive to PAT
Time to set up sample None. Just turn it on. Can be significant. Conventional method requires higher labor costs and time delays.
Human intervention Minimal. Required to set up samples, transport, and to read results. Human intervention creates more possibilities for inaccuracies.
Cost per test Limited to maintenance of device and low cost disposables. Can be significant. Instant Microbial Detection method lowers per test cost.
Table 1: A Comparison of Current Method vs. Proposed Method

(III) To Verify If Remediation Was Successful
Finally, the IMD devices can be used to obtain immediate results, for example, after remediation of microbial contamination has taken place, to determine,on a real-time basis, if the remediation was successful.

A Comparison of Current Method vs. Proposed Method
The table above compares the Instant Microbial Detection method against thecurrent plate culture method.

Summary
The IMD-A and IMD-L devices provide instantaneous and continuous microbial detection, which make the devices extremely useful tools for pharmaceuticalmanufacturers to:

  • Implement PAT;
  • Comply with both regulatory and internal requirements for microbial monitoring and remediation;
  • Significantly reduce costly time delays and product contamination.

Reference:

1. Mie scattering is an optical phenomenon where a beam of light is scattered by particles whose sizes are comparable to the wavelength of the light. In this case, the scattered light intensity is dependent upon the particle size. Using this principle, one can determine the sizes of particles by measuring the light intensity scattered by those particles.

Chuck Bolotin is Vice president of Sales at BioVigilant, 2015 W. Ruthrauff Road, Suite 153, Tucson, AZ 85705. He can be reached at 520-292-2342 or Chuck.Bolotin@BioVigilant.www.biovigilant.com

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