Monitorización ambiental

El Procedimiento de buenas prácticas de fabricación (GMP) establece las condiciones de calidad del aire para la producción biofarmacéutica en salas limpias. El peligro real son los microbios del cuerpo humano. Los seres humanos arrojan alrededor de 30 000 células cutáneas por hora3, todas las cuales son potenciales portadoras de microbios. Lamentablemente, no disponemos de tecnología para detectar microbios en el aire en tiempo real, por lo que se usan contadores de partículas en el aire como sustitutivo. 1

Clases/Grados de limpieza

Las BPF de la UE y las BPFa de los EE. UU. definen el recuento de partículas en el aire/m³ para cada clase/grado de limpieza.2 Existen diferencias fundamentales entre los documentos de BPFa de EE. UU. y BPF de la UE para las clases/grados de sala limpia. La diferencia más destacada es que las BPF de la UE requieren que el usuario de la sala limpia clasifique y supervise posteriormente sus salas limpias para partículas en el aire de ≥0,5 micras de tamaño y también ≥5 micras de tamaño, mientras que el BPFa de EE. UU. solo requiere que el usuario clasifique y posteriormente monitorice sus salas limpias para partículas en el aire de ≥0,5 micras solamente.

Clasificación de área limpia (partículas de 0,5 µm/pie)3) Designación ISO Partículas de ≥0,5 µm/m³ Niveles de acción de aire activo microbiológico (cfu/m3) Niveles de acción de las placas de sedimentación microbiológica (diám. 90 mm; ufc/4 horas)
100 5 3520 1 1
1000 6 35 200 7 3
10 000 7 352 000 10 5
100 000 8 3 520 000 100 50

Figura 1. Clases de sala limpia según las BPFa de EE. UU.

 

Número máximo de partículas permitido por m3 mayor o igual que el tamaño tabulado
En reposo En funcionamiento
Grado 0,5 µm 5,0 µm 0,5 µm 5,0 µm
A 3520 20 3520 20
B 3520 29 352 000 2900
C 352 000 2900 3 520 000 29 000
D 3 520 000 29 000 Sin definir Sin definir

Figura 2. Grados de sala limpia según se define en las BPF de la UE

Como también se puede ver en las tablas de las Figuras 1 y 2, las BPFa no definen los niveles de limpieza en el aire para cada clase de sala limpia cuando la sala limpia está en reposo, mientras que las BPF diferencian entre estos dos estados. La tercera diferencia importante entre los dos documentos es que las BPFa tienen una clase adicional entre clase 100 (equivalente a grado BPF grado A) y clase 10.000 (equivalente a BPF grado A en funcionamiento) y las BPF tienen un grado D adicional, definido como ≤3.520.000 partículas de ≥0,5 micras en reposo.

 

Referencias

  1. Harrison, Tony. “Automating Biopharma Quality Control to Reduce Costs and Improve Data Integrity.” Beckman Coulter Life Sciences. Page 3, PART-3506WP02.18 February, 2018.
  2. Harrison, Tony. “Changes to GMP force cleanroom re-classifications.” Beckman Coulter Life Sciences. Page 1, PART -1352WP02.16-A. February 2006.
  3. Health How Stuff Works, How many skin cells do you shed every day?, by Ed Grabianowski. http://health.howstuffworks.com/skin-care/information/anatomy/shed-skin-cells.htm, published July 6, 2010.

Cleanroom types

By its simplest definition, a cleanroom is a contained space where effort is put toward limiting the presence of unwanted particulates, as well as keeping other environmental conditions such as temperature, humidity and pressure optimized to the work being done. 

Across many industries—aerospace, semiconductor manufacturing, military, medical devices, biotech and pharmaceuticals to name a few—cleanrooms are essential to any manufacturing operation where reducing contamination from airborne particles is critical to success.

How important have cleanrooms been to industry? Within only a few years following their initial development in 1960, cleanroom technology expenditures worldwide had already reached the modern equivalent of US $400 billion. Cleanrooms have evolved since then, varying widely in size and complexity, but they still fall into two main categories based on how the air flowing through them is handled:

Turbulently ventilated cleanrooms 

Also known as nonunidirectional cleanrooms, turbulently ventilated cleanrooms are most often designed to receive clean filtered air from the ceiling through multiple air diffusers (although systems using a single non-diffusing source inlet do exist). Incoming air then mixes with room air as it moves along a fairly unpredictable path, picking up particulate airborne contaminants and finally leaving the room through returns located in the walls at floor level. 

cleanroom monitoring

The turbulently ventilated design is most effective for removing larger particulate contaminants such as dust from the air. As such, it’s a system that may be suitable for certain pharmaceutical compounding and packaging applications, but is insufficient for more sensitive biopharmaceutical operations, where the presence of smaller microbial contaminants such as bacteria and viruses can be disastrous (see Classifications & Standards below). 

The slower, more circuitous airflow within nonunidirectional cleanrooms leads to another shortcoming: various areas of the room may, for a period of time, “hold onto” pockets of air with low circulation and high particle counts. These are a result of air currents, most often created by the movements of operators and placement of equipment, combined with the unpredictable nature of downward airflow characteristic of this cleanroom type.

Unidirectional airflow cleanrooms

Also known as a laminar flow cleanroom, this is the cleaner of the two types—at the expense of requiring a great deal more air to operate. Rather than air diffusers, this setup uses laminar airflow hoods that typically cover anywhere from 35% – 100% of the ceiling space. From these, jets of filtered air are sent straight downward at a constant velocity in streamlines that are nearly parallel to one another.

Floors in this type of cleanroom are typically constructed with raised tiles, perforated throughout to ensure that air can travel along a completely unimpeded path from entrance to exit. After leaving the room, particulates picked up in the airstream are either trapped with vacuum pumps beneath the floor, or removed by the ceiling filters when the air is sent back for recirculation.

cleanroom environmental monitoring high efficiency filters

Compared with turbulently ventilated arrangements, unidirectional airflow systems greatly reduce the chance of airborne contaminants moving around the space at random and eventually settling where they shouldn’t be. With parallel airstreams flushing the room from top to bottom, there’s little risk of low-circulation, high-particle-count pockets developing as they invariably do with turbulent, nonunidirectional airflow. Any potential for localized turbulence in laminar flow cleanrooms is further mitigated by the architecture of the room itself—i.e., free of unnecessary features that could become obstacles to smooth airflow.

Larger cleanroom facilities sometimes employ mixed flow ventilation as a way of providing unidirectional airflow to certain ultra-clean areas that require it, without the greater expense of designing the entire room this way. Other areas in the same room requiring less stringent conditions of cleanliness can be outfitted with nonunidirectional-style diffusers and extracts where more turbulent airflow won’t interfere with the cleaner laminar flow locations.

Filtration in cleanrooms

No matter the type of airflow mechanism used, the real power of any cleanroom is in its capacity for filtering the air that moves through it. At minimum, all air delivered to a typical cleanroom passes through ceiling-installed high-efficiency particulate air (HEPA) filters that trap particles ≥ 0.3 µm in size with up to 99.97% efficiency. For operations requiring the highest levels of cleanliness, ultra-low particulate air filters (ULPA, trapping ≥ 0.1 µm particles with up to 99.9995% efficiency) may be used.

It’s easy to visualize airborne particulates and microbial contaminants larger than these threshold sizes being stopped by a simple filter mesh while smaller particles pass through it, yet the actual filtering mechanics are more complex. Particle filtration relies on four main principles: inertial impaction, interception, diffusion, and electrostatic attraction.

  • Impaction occurs when a large particle (usually ≥ 10 µm) with high inertia veers from the air stream and collides with a filter fiber the stream is moving past.
  • Interception occurs when a large particle (1.0 - 10 µm) stays within the airstream, but collides with and sticks to a fiber as the stream moves through that fiber.
  • Diffusion affects particles small enough (≤ 1.0 µm) to exhibit Brownian (random) motion. The vibrational movement they naturally acquire gives them a high probability of contacting a fiber and being removed from the airstream. Diffusion is the primary means by which HEPA and ULPA filters remove small particles from the air entering a cleanroom.
  • Electrostatic attraction increases filtering efficiency by allowing the capture of charged particles moving through the airstream. 

Other considerations

Beyond the concerns of airflow and air filtering are several other structural considerations that must be addressed if a cleanroom is to function efficiently and reliably, including:
  • Airlocks (also called anterooms) are the spaces connecting cleanrooms with rooms of lower cleanliness levels. Airlocks can help maintain a negative pressure differential that keeps air and particulates from flowing toward the cleaner areas, as well as provide a space to unpackage equipment before it is taken into the cleanroom.
  • Windows are almost always needed in cleanrooms for observation. They must be made from impact-resistant glass and installed so as to minimize any non-aerodynamic ledges or other protrusions into the cleanroom interior.
  • Siting of a cleanroom can be critical to efficient operation. Factors such as the movements of entering and exiting personnel and vibrations from equipment must be taken into account when deciding where a cleanroom will be situated. Even the running motors from a nearby parking lot can create enough vibration to jar loose particles within a cleanroom. More severe vibration from close mechanical sources can encourage the appearance of leaks in ductwork and filters. 

Cleanroom standards for biopharmaceutical operations

In biopharmaceutical manufacturing, the potential for ruinous contamination by living microorganisms during processing is an ongoing concern. And, due to the biomolecular nature of the products made in such facilities, it’s the operators themselves who are the chief source. Humans shed around 35,000 skin cells per hour on average2, all of which are potential carriers of contaminating microbes.

Common skin-associated bacteria include Micrococcus, Staphylococcus, Corynebacterium and Bacillus. Contamination by bacterial species from these groups and others can ultimately have an enormous impact on biopharmaceutical product manufacture. At the least, their presence can change product consistency and impurity profiles. Bacterial contaminants can also cause potency loss through enzymatic modification or degradation, and can impart secreted bacterial endotoxins to the product.

In addition, it has been found that fungal species from genera Aurebasidium, Cladosporium, Aspergillus and Pencillium are the most commonly isolated from cleanrooms, with the latter two being the most prevalent found on the human body along with Malassezia spp. Fungal contamination by these organisms can result in spoilage of pharmaceutical products and possibly pose more serious health hazards to those taking them, particularly immunocompromised patients. 

To help keep these microbiological sources of contamination in check, cleanroom operators are outfitted with a garment system (boots, gloves, hood, facemask) to provide a barrier between the wearer and the controlled work environment. However, no garment system can ever be 100% effective. 

Given that our outer layer of skin can host as many as 1 million microorganisms per square centimeter—20 billion across the average human body—microbes will continue to be isolated from cleanrooms, even of the more stringent classifications, and where personnel are properly gowned, wearing masks and full body covers, and where aseptic technique is diligently practiced.

In terms of countering bacterial and viral agents, lower-stringency cleanrooms may be sufficient for production of medical compounds meant for oral administration (capsules, tablets and liquids) because, when these are ingested, the gastrointestinal tract generally kills any stray microorganisms ingested along with them. However, parenterals (medicines designed for injection into veins or muscles) enter the bloodstream without these protections and must be produced under a far higher standard of cleanliness. Airborne microbial particulates entering the product must be avoided at all costs. Sterile conditions over a large area are required for parenteral processing operations, and cleanrooms make this possible.

Because we know that microbes do still enter the cleanroom airstream, their presence and concentrations must be known, so that any emerging contamination concerns can be addressed, and a cleanroom’s operational efficiency can be accurately evaluated. The technology doesn’t yet exist to track airborne microbes in real time, so high-precision particle counters are used to detect them indirectly. They do this by sampling a cleanroom environment and providing data on the number of particulates (including microorganisms) within a given size range that occupy a given volume of air. This is where cleanroom classification comes in.

Cleanroom designations

Cleanroom designations in the US were originally given names based on the maximum number of particles of 0.5 µm or larger allowed in a cubic foot of space—Class 1000, Class 10,000 and so on—as defined by the FDA Federal Standard 209E. 

While this standard is still in practical use, the International Organization for Standardization (ISO) published its ISO 14644 document series in 2001 to define the "standardization of equipment, facilities, and operational methods for cleanrooms and associated controlled environments." This new standard set was quickly adopted in Europe and officially superseded FED STD 209E, converting imperial units to metric and defining simpler ISO classes according to the familiar particle-based criteria. For example, what was once called a Class 1000 cleanroom has become an ISO Class 6, allowing a ≥ 0.5 µm particle count of 35,200/m3.

The partial table below shows equivalences between the older US classifications (leftmost column) and the newer ISO designations used for some of the most common cleanrooms found in the biopharmaceutical industry. 

In terms of cleanliness, rooms rated ISO 8, ISO 7 and ISO 6 are considered “non-stringent” or “intermediate” cleanrooms, sufficient for many pharmaceutical applications. Those designated ISO 5 and below are “stringent” cleanrooms, almost invariably featuring laminar airflow and HEPA-filter ceiling coverage approaching 100 percent. The cleanest of these (ISO 2 and ISO 1) are often required for highly sensitive electronics manufacturing. There is also an ISO 9 designation—the “dirtiest cleanroom”—descriptive of a minimally controlled office space where up to 35.2 million particles/m3 can be expected. 

Clean Area Classification (0.5µm particles/ft3) ISO Designation ≥0.5µm particles/m3 Microbiological Active Air Action Levels (cfu/m3) Microbiological Settling Plates Action Levels (diam. 90mm; cfu/4 hours)
100 5 3,520 1 1
1000 6 35,200 7 3
10,000 7 352,000 10 5
100,000 8 3,520,000 100 50

Figure 1. Cleanroom classes as defined in USA cGMP

Over the years, several clarifying additions have been made to the ISO document series but the guiding principles stay the same. Although cleanrooms are now categorized for particle sizes as low as 0.1 µm, biopharmaceutical facilities are still chiefly concerned with those 0.5 µm and larger. This is because microbes such as bacteria and viruses are only rarely found in the air as single units and are far more typically attached to larger airborne particles, such as skin cells, in a size range of 10 to 15 µm.

Adding a level of complexity (and for reasons not clearly understood) there are still significant differences between cleanroom standards applied in the US and those used by European Union nations, where a letter grading convention is used in place of the FED STD 209 numbering system:

Maximum permitted number of particles per m3 equal to or greater than the tabulated size
At rest In operation
Grade 0.5µm 5.0µm 0.5µm 5.0µm
A 3,520 20 3,520 20
B 3,520 29 352,000 2,900
C 352,000 2,900 3,520,000 29,000
D 3,520,000 29,000 Not defined Not defined

Figure 2. Cleanroom grades as defined by EU GMP

Current US Good Manufacturing Practice (US cGMP) and EU GMP regulations both require operators to classify and subsequently monitor their cleanrooms for airborne particles according to similar methodologies; however, Figure 2 above highlights a few important differences to keep in mind:

  • While US cGMP and EU GMP both require classification and monitoring of cleanrooms for airborne particles ≥ 0.5 µm in size, EU GMP additionally requires the same attention to airborne particles ≥ 5 µm—as mentioned above, a particle size especially relevant to biopharmaceutical operations.
  • While US cGMP defines airborne cleanliness levels only for cleanrooms that are in operation, EU GMP adds a separate set of particle count maximums that apply when a cleanroom is at rest. 
  • The US cGMP includes an additional class designation (1,000) found between the EU GMP-equivalent Grades A and B.
  • The EU GMP includes an additional Grade D for at-rest cleanrooms, defined by a maximum particle count of ≤ 3,520,000 in the ≥ 0.5 µm size range. 

Safety first—and always

Particularly in the biopharmaceutical industry, economic survival absolutely depends on the safety of the finished product. The possibility of contamination compromises that safety, making it crucial that manufacturers familiarize themselves with:
  • The different types and designations of cleanrooms used in the industry
  • The most common sources of manufacturing process contamination that cleanrooms are designed to prevent
  • All Good Manufacturing Practice (GMP) standards regulated and enforced by applicable government agencies 
  • The need for high-precision environmental monitoring equipment used to ensure GMP compliance, consumer safety, and company health

It should be clear why the cleanroom is an essential component to any biopharmaceutical manufacturing operation—and why maintaining the integrity of the cleanroom environment can’t be left up to guesswork, any more than the pharmaceutical development and testing process itself.

Productos para la monitorización ambiental

Facility Monitoring System

Sistemas de vigilancia de instalaciones (FMS)

Soluciones escalables de monitorización de partículas en línea basadas en comunicaciones de arquitectura abierta que se integran fácilmente y permiten el cumplimiento del Anexo 1 de las GMP de la UE, la FDA y su sistema de calidad interno. 

MET ONE 3400

Contador de partículas en el aire portátil para la monitorización ambiental de salas limpias

MET ONE 6000

Contador de partículas en el aire en línea con sensibilidad de 0,3/0,5 μm diseñado para el cumplimiento de la norma ISO 21501

Contadores de partículas de mano serie MET ONE HHPC+

La serie MET ONE HHPC+ de contadores de partículas de mano es ligera a la hora de comprobar la validación ISO Clase 5 (FED STED Clase 100) o salas limpias y entornos controlados de nivel superior. Cuenta con una pantalla grande y una descarga de datos sencilla mediante un cable USB, una llave de memoria o una conexión Ethernet. 
Enlaces útiles

Recursos de aplicación

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