Historically, capture and subsequent culture of microorganisms has been the only technique available for accessing airborne contamination in cleanrooms and clean spaces. Like any analytical method this approach has fundamental limitations. For certain applications, such as in-process monitoring inside restricted Grade A spaces, growth-based methods present challenges to safe and efficient aseptic processing. Firstly, media dries out and must be replaced which requires interventions; secondly, the culture data are retrospective. Interventions introduce significant risk to product sterility while retrospective data does not allow for immediate corrective action. Laser induced fluorescence, or LIF, is an airborne microbial detection technique that has been in use for military threat detection for decades. With the introduction of purpose-built LIF instrumentation, the technology is now poised to impact aseptic processing by overcoming shortcomings of traditional techniques. This article will outline how LIF functions and specific ways LIF can benefit biopharmaceutical manufacturing.
Principles of Real-Time Viable Particle Discrimination: Laser Induced Fluorescence
Laser induced fluorescence (LIF) is sometimes referred to as “bio-flourescent particle counting”, or “autofluorescent particle counting”. All of these names refer to the same core technology, which more closely resembles optical particle counting than traditional microbiology. In LIF, each airborne particle is interrogated independently and a deter-mination is made whether an individual particle has characteristics consistent with a microorganism or benign airborne particulate.
To discuss LIF it is first necessary to have a basic understanding of optical particle counting. This is the ubiquitous cleanroom monitoring technology used to determine the concentration and size distribution of total airborne particles (generally >500 nm). Particles are sampled and directed through an optics chamber where they pass through a laser beam; typically a red laser. Each particle scatters the laser light as it passes through the beam. Light-scattering events are enumerated and reported with respect to the volume of air sampled, i.e. number of particles per volume of air. Moreover, the intensity of each light scattering event is recorded and this information correlates to the size of the particle, a phenomenon commonly referred to as Mie scattering. Thus, the optical particle counter reports the concentration and size distribution of airborne particles.
Like optical particle counting, LIF also employs laser light. To determine whether an airborne particle is viable or non-viable (i.e. a microorganism or not), LIF leverages the fact that microorganisms contain relatively high concentrations of fluorescent molecules. Table 1 highlights select molecules which can contribute to microbiological autofluorescence. When laser light impinges on a microorganism not only is light scattered but some of the light is absorbed and reemitted at higher wavelengths. This fluorescent light is distinct from Mie scattered light which occurs at essentially the same wavelength as the impingent laser light. As depicted in Figure 1, the intensity of fluorescent light is a critical characteristic for the discrimination of viable particles by LIF.
Table 1. Autofluorescent molecules present in microorganisms.
Figure 1. Simplified representation of the optical characteristics of viable and non-viable particles.When a microorganism enters the LIF optics chamber it transits the beam of a blue laser (blue light better matches the average absorbance of the various fluorescent molecules in microorganisms). As depicted in Figure 1 on the right (VIABLE), two distinct optical phenomena occur. Some of the laser light is absorbed by fluorescent molecules and reemitted at higher wavelengths and some of the light is scattered. Both the scattered light and the fluorescent light are focused and filtered, before their intensities are detected. The intensity data is used to determine if the particle is viable or non-viable.
How this data is collected and how it is used to discriminate viability differs between instrument manufacturers. For example, in the BioTrak® Real-Time Viable Particle Counter model 9510-BD from TSI Inc., three different light intensities are measured for each particle; scattered light intensity, fluorescent light intensity from 430 to 500 nm, and fluorescent light intensity from 500-650 nm (excitation occurs at 405 nm). The three parameters are processed by a proprietary algorithm and a viability determination is made. The algorithm was derived empirically through the measurement of numerous microorganisms and inert particles. Figure 2 shows the value of measuring multiple optical parameters for discrimination. Panel A shows pollen and bacteria poorly discriminated by two parameters, panel B shows three optical parameters (as measured by BioTrak model 9510-BD) providing discrimination between common viable (bacteria) and non-viable (pollen) particles.
Figure 2. Improved viable discrimination using three optical parameters. Panel A only shows two optical parameters, resulting in poor discrimination. Panel B shows markedly improved discrimination using three optical parameters. Both panels show superimposed data from Ralstonia Pickettii (red dots) and Bermuda Grass pollen (blue dots), collected on BioTrak Particle Counter model 9510-BD (TSI Inc.).Because optical analysis is non-destructive, total particle counting and viability detection can be combined with each other and/or other devices. For example the above referred BioTrak Particle Counter model 9510-BD instrument combines total particle counting and viability detection. It contains two optics chambers, one for total particle counting and another for viability discrimination. The unit also captures particles on a gelatin filter device after optical analyses in order to support subsequent growth for identification and/or confirmation of positive results.
Where LIF Excels Compared to Growth-Based Methods
Microbiological techniques for environmental monitoring rely on capturing airborne microorganisms, providing conditions permissive for expansion and subsequent detection of the resulting colony. Although many growth-based products and instrument are available, this approach has limited flexibility. LIF offers some distinct advantages which add value and improve process knowledge for aseptic manufacturing.
Real-Time Data: The most obvious limitation of traditional microbial monitoring is the time required for culture. The longer the period between sample collection and detection of contamination the less actionable the information becomes. LIF-based methods offer “real-time” data. Data can be collected on the order of seconds or minutes depending on the configuration and operation of the instrument.
No Media = No Dehydration: Since LIF does not require capture and culture for microbial detection, regular delivery of fresh media to the sample is not required. Moreover, the unit can physically reside outside of restricted Grade A areas such as isolators and RABS, in much the same configuration as optical particle counters. In these restricted spaces, line stoppages for in-process microbial monitoring can be eliminated by implementing LIF-based detection methods.
Continuous Monitoring: Some industry experts speculate that future regulatory expectations may include continuous, in-process microbial monitoring. Using traditional methods it is very difficult to achieve truly continuous microbial monitoring. LIF allows continuous sampling for days, weeks, even months. This type of data contributes to process understanding and allows operators to know the process is in control before, after and throughout the campaign.
Temporal Resolution: Most traditional growth-based methods cannot deliver time-resolved data. Those methods that do offer some degree of temporal resolution - slit-to-agar samplers, for example, cannot deliver the result fast enough to be actionable. Real-time LIF results are sufficiently fast and time-resolved for the data to be immediately actionable and allow manufacturers to confidently associate excursions with specific events.
Manufacturing Benefits of In-Process, LIF-Based Monitoring During Fill-Finish
One application where LIF-based methods show huge potential for real-world manufacturing improvement is replacing traditional growth-based methods for in-process monitoring during aseptic fill-finish processes. In this setting LIF offers real-time process knowledge, continuous and interruption-free operation, and automated data collection and analysis with maximum data integrity. Importantly, the elimination of traditional growth-based methods can introduce significant business benefits for the manufacturing facility.
Increase Yield
Reduce wasted drug product: For in-process sampling, agar plate changes require process interruptions. One risk mitigation strategy following production process interruptions is wasting filled drug containers exposed during the intervention period. This practice achieves effective risk mitigation but sacrifices throughput. LIF does not require interventions for in-process data collection.
Furthermore, many biologics are formulated at high concentration and therefore present a challenge for automated fluid handling. One challenge is dehydration and solidification at the exposed nozzle tip. This process is greatly exacerbated during periods of stoppage that can result in wasted drug product. LIF’s ability to sample continuously without interruption can reduce fluid handing challenges associated with difficult formulations.
Increased capacity: Production interruptions for microbial mon-itoring can add up to days even weeks over the course of year. With LIF-based methods much of this valuable production time is recovered, shortening batch processing times and freeing capacity for additional batches.
Reduction in Cost of Goods Manufactured (COGM)
Reduced downtime: Labor and facility-dependent manufacturing costs can account for >70% of COGM (excluding API costs). Depending on the frequency and duration of microbial monitoring interventions, these operations can consume 20% of production time. LIF-based detection can eliminate downtime to perform microbial monitoring and reduce COGM accordingly.
Reduced microbiology costs: One large pharmaceutical firm estimates the actual cost of a single, validated settle to be between $60 and $100. That figure considers the entire process and includes costs such as growth promotion, LIMS, FTE time, culture equipment and space, etc. With LIF-based monitoring there is no need to process thousands of plates annually to perform in-process microbial monitoring potentially saving millions of dollars all while improving product safety.
Summary & Conclusions
Laser Induced Fluorescence (LIF), also called “bioflourescent particle counting” or “autofluorescent particle counting”, is an alternative microbiological method for the detection of airborne microorganisms. LIF is not growth-based. Rather, it detects individual microorganisms in real-time, giving it the flexibility to improve aseptic manufacturing processes. By eliminating interventions for agar plate changes in the aseptic core and by providing real-time, continuous data, LIF has the potential to reduce risk while increasing manufacturing efficiency and capacity.
References
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