In 2026, the M3 Collaboration is launching a structured, monthly blog series focused entirely on

biofluorescent particle counting (BFPC). Rather than offering a singular perspective, we will

examine the topic progressively across the year. BFPC technology represents a shift in how viable

(air and water) monitoring data can be generated and used, and that shift is important enough to

warrant deeper explanation and practical context. Our objective is to create a technically grounded

resource that breaks the technology into usable components, what it measures, what the data

means, and how it fits within established contamination control strategies. This series is designed

to move beyond awareness and into practical understanding while still making the content

accessible to everyone.

For those unfamiliar with the initiative, the M3 Collaboration is a volunteer organization that was

formed in 2021 by several industry groups coming together and aligning around advancing modern

microbiological methods. These groups joined forces to support practical, science-based

implementation of alternative and rapid microbiological methods across pharmaceutical,

biopharmaceutical, medical device, personal care, and vendor sectors. The collaboration brings

together subject matter experts across microbiology, quality, engineering, and regulatory

compliance, and that cross-functional perspective continues to shape the output and discussions

for this group throughout the year.

Across the blog series, we will move through the questions that we believe matter most to facilities

evaluating or implementing BFPC technology. We will begin with fundamentals: what BFPCs

detect, what real-time viable particle data represents, and how that differs from the traditional

colony-forming unit (CFU) recovery. From there, we will examine application: where BFPCs add

value in aseptic environments, how placement should be considered in critical zones, and what

representative monitoring means when viable data is continuous rather than periodic.

We will then address integration and mechanism together. A monitoring technology has

tremendous value when it strengthens the broader contamination control strategy, and we are able

to demonstrate this effectively to broader leadership. We will discuss how continuous viable

particle data can provide earlier visibility into atypical events and support more focused

investigations. At a high level, we will also explain the scientific basis of laser-induced

fluorescence and how available systems differentiate biological particles from inert ones,

providing sufficient technical grounding for an informed, public interpretation.

As the blog progresses later in the year, emphasis will shift towards interpretation and

implementation. We will introduce what auto-fluorescent units (AFUs) represent, why they should

not be expected to correlate directly with CFUs, and how to evaluate trends, transient spikes, and potential non-biologic fluorescent particles in the environment. Topics will likely include non-

equivalence, alert strategies, facility-specific baselining, validation expectations, and digital data

management, recognizing that continuous monitoring produces datasets that must be reviewed,

trended, and documented in an inspection-ready manner.

Towards the fall of 2026, this blog will explore equivalence discussions in Grade A environments,

evolving regulatory perspectives, and what real-world deployment looks like. Implementation

requires more than instrument installation; it involves training, change management, cross-

functional alignment, and clear communication of expectations. We will address common

challenges observed in routine practice and practical approaches to managing them.

When relevant, we will link to peer-reviewed publications and broader industry guidance,

including work developed by members of the M3 collaboration and related working groups. The

BFPC subteam within M3 includes individuals with direct implementation and validation

experience, and that operational depth will inform the discussion. The intent is to provide

experience-based insight alongside published science that helps to guide practical, facility

application.

We will publish each installment monthly on both the M3 website and LinkedIn. Our goals are

transparent: maintain an ongoing technical resource, increase visibility of the collaboration,

expand understanding of BFPC technology, and make a complex subject easier to navigate without

losing scientific integrity.

We also encourage active participation. If you have questions, practical challenges, or perspectives

from your own implementation efforts, we invite you to share them in the comments or submit

them for future discussion. Strengthening the responsible application of BFPC technology will

require open scientific dialogue. Through shared experience and thoughtful exchange, our

community can advance understanding and improve how these tools are applied in practice.

We invite you to follow the series throughout 2026, and if you follow the M3 Collaboration on

LinkedIn, please enable notifications so you receive an alert when each new blog is released.

By Vanessa Figueroa, Petra Merker, and Jessica MacGregor │ March 3, 2026

Environmental monitoring in aseptic processing is evolving. Biofluorescent Particle Counters (BFPCs) are

non-growth-based environmental monitoring systems that have now been implemented in select

pharmaceutical companies, with integrated use particularly in advanced aseptic barrier systems such as

robotic, gloveless isolators. Rather than relying on microbial growth after incubation, BFPCs provide

continuous, real-time detection of biologically derived airborne particles during aseptic processing. This

shifts viable monitoring closer to the process itself and supports active oversight rather than serving solely

as a retrospective, broad verification activity.

To provide context for this shift, this blog offers a practical orientation to the technology. It explains what

BFPCs are, what they measure, and how they differ from traditional culture-based air monitoring. It also

introduces real-time viable particle detection in straightforward terms and clarifies why fluorescence-based

results represent a different type of information than growth-based data.

To understand that difference, it is helpful to recall how traditional viable air monitoring works. Traditional

monitoring relies on growth-based methods in which active air samplers draw a defined volume of air across

an agar surface. After incubation, microorganisms capable of growth form visible colonies that are counted

as colony forming units (CFU). The primary limitation of this approach is that results are retrospective and

available only after incubation and enumeration. In addition, only organisms able to grow under the selected

media and incubation conditions are detected.

BFPC technology approaches detection differently. Airborne particles pass through the instrument’s optical

detection chamber, where they are assessed for light-scattering properties to determine particle size and

total count, as well as for intrinsic fluorescence signals associated with biological material. Certain naturally

occurring cellular components, such as NADH, riboflavin, and other flavins, emit characteristic

fluorescence when excited. By detecting this signal, the system classifies and enumerates particles with

biological characteristics and provides that signal in real time rather than waiting for growth.

BFPC results are typically expressed as auto fluorescence units (AFUs) or fluorescence-based viable

particle counts (VPCs), not CFUs. Because the detection mechanism differs fundamentally from growth-

based methods, these signals are not directly comparable to traditional CFU results. Colony forming units

quantify organisms that have grown and formed visible colonies under defined laboratory conditions,

whereas BFPC outputs quantify particles exhibiting fluorescence characteristics consistent with biological

material at the moment of detection. The two measurements therefore reflect related but non-equivalent

aspects of airborne contamination.

Beyond the detection principle itself, a defining characteristic of BFPC systems is continuous monitoring

throughout critical aseptic processing. This enables ongoing surveillance of the Grade A environment while

reducing manual interventions associated with agar plate changes and generating a far larger volume of

data than traditional discrete sampling. The resulting data set allows trends, transient events, and shifts in

environmental conditions to be observed in near real time rather than interpreted days later. Because

detection is not growth-dependent, BFPC systems may also register viable but non-culturable (VNBC)

particles that may not form colonies under standard culture conditions.

From a regulatory perspective, most guidance documents now recognize the use of alternative and rapid

monitoring technologies. EU GMP Annex 1 encourages consideration of rapid or alternative methods and

continuous monitoring systems in aseptic processing, while requiring scientific justification when results

are presented in a format different from CFU. Fluorescence-based outputs should therefore not be viewed

as a direct numerical replacement for CFU counts, but as a distinct data set requiring appropriate technical

interpretation.

In practical terms, these differences in detection principles translate into differences in process control.

Traditional viable air monitoring confirms the presence of cultivable microorganisms only after incubation,

when processing has already concluded. BFPC systems provide immediate indication of particles with

biological fluorescence during processing, enabling earlier awareness of potential contamination events.

This real-time visibility strengthens aseptic process control by enabling prompt investigation and targeted

response and, where appropriate, segregation or rejection of only the affected portion of a batch rather than

reliance on broader retrospective product impact assumptions.

When appropriately qualified and incorporated into a site’s contamination control strategy, BFPCs provide

continuous insight into airborne biological particles within advanced aseptic barrier systems. The value of

that data, however, depends on thoughtful implementation. Instrument placement, defining representative

monitoring in a continuous data environment, and aligning the sampling strategy with process risk are

critical considerations. These topics will be the focus of the next blog in this series. Stay tuned for more.

References:

Barensteiner, Ruth, et al. Bio-Fluorescent Particle Counter (BFPC) Continuous Environmental Viable

Particle Monitoring Strategy for Aseptic Filling. ISPE D/A/CH Affiliate Future Robotics SIG, Feb. 2023.

EudraLex. EudraLex Volume 4: EU Guidelines for Good Manufacturing Practice for Medicinal Products

for Human and Veterinary Use, Annex 1: Manufacture of Sterile Medicinal Products. European

Commission, Aug. 2022.

Hutchins, Patrick M. “Real-Time Viable Particle Monitoring: Principles and Benefits for In-Process

Measurements.” American Pharmaceutical Review, 1 Dec. 2016.

Krebsbach, Timo, et. al. “Environmental Monitoring in Aseptic Manufacturing.” Pharmind 88, Nr.2, 146-

153, 2026.

Merker, Petra. Quality Risk Management and Implementation of Continuous Real-Time Environmental

Monitoring in Aseptic Processing. 2024.

Scott, Allison, et al. “Challenges Encountered in the Implementation of Bio-Fluorescent Particle Counting

Systems as a Routine Microbial Monitoring Tool.” PDA Journal of Pharmaceutical Science and

Technology, 2022, doi:10.5731/pdajpst.2021.012726.

Contributing Authors: Petra Merker, Noel Long, and Vanessa Figueroa│ April 15, 2026

This blog is presented by members of the M3 collaboration. The opinions expressed may not be fully

representative of all the individuals in the collaboration or their respective employers.

In the previous blog, we introduced Biofluorescent Particle Counters (BFPCs) and explained how they

differ from traditional culture-based viable air monitoring. The next practical question many organizations

ask is where these systems should be placed within an aseptic environment.

Placement is not simply a matter of installing sensors near the product path. As with any environmental

monitoring program, sampling locations must be selected through a structured, risk-based evaluation that

reflects the process, the equipment design, and the potential routes by which contamination could reach the

product. At the same time, BFPCs introduce continuous monitoring and real-time data, which changes how

the concept of representative monitoring is interpreted.

This blog explores where BFPCs can add value in aseptic processing environments, key considerations

when determining monitoring locations in critical areas, and how representative monitoring should be

approached when data are collected continuously rather than at discrete sampling intervals.

The selection of BFPC monitoring locations follows the same fundamental approach used for traditional

environmental monitoring methods: sampling points should be defined through a structured risk

assessment. This assessment applies the principles of Quality Risk Management (QRM) and forms part of

the site’s overall Contamination Control Strategy (CCS).

The starting point is a clear understanding of the manufacturing process and the design of the aseptic

environment. Individual sections of the isolator or filling line are evaluated based on their potential

contamination risk. Factors such as product exposure, material flow, equipment design, and the types of

interventions that may occur during processing are considered. Assigning relative risk levels to these

segments helps determine where environmental monitoring will provide the most meaningful information.

BFPC technology is particularly well suited for closed barrier aseptic environments such as closed

Restricted Access Barrier Systems (cRABS), isolators, and increasingly gloveless or highly automated

robotic isolators. In these environments, the product is separated from operators and the surrounding

cleanroom by engineered barriers, and material transfers occur through controlled mechanisms. These

environments are typically biodecontaminated prior to processing to establish a defined starting condition,

and airflow is tightly controlled to protect the critical zone.

Because environmental monitoring programs should reflect the level of containment and automation

present, the number and type of monitoring locations may vary between different aseptic environments.

Highly automated isolators and robotic filling lines may justify fewer monitoring points when supported

by a robust risk assessment and continuous monitoring capability. In contrast, environments with greater

operator interaction, such as some RABS configurations, may require additional monitoring points due to

the increased likelihood of microbial contamination events.

Structured tools can help support the risk assessment used to determine monitoring locations and the

transition from traditional growth-based monitoring methods to alternative technologies such as BFPCs.

For example, the Biophorum guidance document titled “Harmonized Risk-Based Approach to Selecting Monitoring Points and Defining Monitoring Plans” provides a framework for applying QRM principles

when defining environmental monitoring locations.

Monitoring locations should reflect the areas where contamination would have the greatest potential impact

on product exposure. Engineering studies are used to support these risk-based decisions. Computational

fluid dynamics modeling and airflow visualization studies can help characterize airflow patterns and

identify locations where monitoring will best represent aseptic conditions within the critical zone.

Probes should be positioned so they do not disrupt unidirectional airflow or create turbulence that could

alter particle movement within the aseptic environment. Potential sources of background fluorescence

should also be considered during placement. Certain materials present in cleanroom environments,

including disinfectant residues, wipes, and some polymer components, may produce fluorescent signals

that can be detected by the system. When sensors are positioned to the most critical parts of the filling

process while preserving airflow integrity and minimizing interference, the resulting data stream permits

continuous visibility into the state of the environment surrounding the sterile product.

Rather than totally replacing traditional environmental monitoring principles, BFPC technology expands

our understanding by providing real-time insight into airborne biological particles during processing. In the

next blog, we will look more closely at how BFPC data can strengthen a company’s contamination control

strategy by providing earlier visibility into potential contamination events, supporting more robust

investigations, and offering new opportunities to better understand the behavior of the aseptic process.

Ref.: “Harmonized Risk-Based Approach to Selecting Monitoring Points and Defining Monitoring Plan”

Biophorum, 2020.

Contributing Authors: Kai Dirscherl, Kaj Anderson, Chris Knutsen, Kim Perkins │ Sept 3, 2026

This blog is presented by members of the M3 collaboration. The opinions expressed may not be fully

representative of all the individuals in the collaboration or their respective employers.

As for any other measuring instrument, having a bio-fluorescence particle counter (BFPC)

calibrated, cleaned and serviced on a regular basis ensures that the instrument remains fit-for-use

and with a performance comparable to that of its time of purchase and first installation. During

the application of a BFPC, its measurements may drift due to, among other causes, instrumental

wear, handling issues, or accidental internal deposition of sampled material from the

environment that it is set to monitor.

Knowing the actual magnitude of measurement errors of an instrument is even more important

than simply adjusting the measurement errors back to zero. The impact assessment of these

errors on the product quality becomes paramount to ensure a high and constant quality standard,

with the ultimate risk of eventual product recall in case an unacceptable change in a BFPC’s

measurement capability has been detected.

At first glance, the optics and the signal processing of a BFPC resemble those of a traditional

Optical Particle Counter (OPC). They both count particles and analyse information contained in

light measured on an intensity scale, but while an OPC only determines the physical size of the

particle on a dimensional scale, a BFPC’s additional task is to conclude “Yes” or “No” on the

particle’s potential biological viability.

Consequently, gradual drifting of an OPC’s calibration can immediately be interpreted as gradual

errors on the particle’s size measurement as described in standardized calibration methods.1 The

same principal methods are applicable to a BFPC, too.2 However, gradual drifting of a BFPC

cannot be mapped to gradual errors in the viability determination in a straightforward way. First

of all, a binary Yes/No decision on viability is a discrete function and therefore naturally does not

operate with a gradual scale. Secondly, the decision logic of a BFPC typically applies a non-

disclosed signal analysis algorithm, which may not be transparent to the end-user.

Nevertheless, it is still possible to establish a gradual metric for a BFPC’s detection efficiency as

a function of calibration errors. At the beginning of a BFPC’s life-cycle, its viability detection

performance is assessed against a set of viable microorganisms under controlled conditions. This

is typically done either by the manufacturer during the BFPC’s primary validation or at the end-

user’s specific environment, where the recovery rates for the various microorganism are part of the required parameters during specificity testing.

Maintaining the calibrated state of the BFPC during the primary validation is key, and calibration

errors are controlled to be zero. At this opportunity, a series of systematic calibration errors may

be advantageously introduced to the BFPC to investigate how these errors impact the recovery

rates for the different microorganisms. Rather than physically adjusting the technical parameters

of the instrument, a more elegant approach is to record the raw light intensity data of zero error

BFPCs. Afterwards this data can be modified offline in silica to simulate instruments with calibration errors by systematically shifting numerical data as if it had been recorded this way, e.g. by dirty optics. Subsequent processing of the modified raw data with the detection algorithm and recording of changes in the microbial recovery rates allow to map percentual changes in the calibration errors to percentual changes in the recovery rates. Both options, physical adjustment of calibration settings or software simulation of misaligned measurement data, require a high level of technical knowledge, and can typically only be performed by the BFPC manufacturer.

Future calibrations benefit from such a mapping of the physical parameter space to the biological

recovery rate with these achievements:

1) In case all physical calibration errors of a BFPC are found to be zero, the BFPC can be

considered to perform equivalently to its primary validation. We have thus established a

reference point, and the actual biological detection efficiency of the BFPC can be

expressed relative to its performance at primary validation, which can be set to 100 %.

2) In case that one or more calibration errors are found to be different from zero, their

impact on the biologic detection efficiency can be easily assessed and expressed in

percent. This not only allows reviewing possible performance changes of the BFPC but

also facilitates the establishment of accept criteria for a maximum permitted percentual

loss in the detection efficiency.

Now this biological detection efficiency is a new metric which links the physical calibration

results to the performance at the primary validation. This information can then be applied by the

end-user for quality assurance, such as 95% confidence intervals of detection results, or trending

of historical instrumental drift.

References

1. International Organization for Standardization. (2018). Light scattering airborne particle

counter for clean spaces, ISO 21501-4:2018

2. Lieberherr, G. et al.: Assessment of real-time bioaerosol particle counters using reference

chamber experiments, Atmos. Meas. Tech., 14, 7693–7706, https://doi.org/10.5194/amt-

14-7693-2021, 2021.