To develop most parenteral drugs, specifically biological products, aseptic manufacturing is a must, but this process can also risk introducing contaminants at various points of the manufacturing process.
There are stringent guidelines and regulations put forward by the US Food and Drug Administration (FDA) and European regulatory bodies to ensure proper control of aseptic cleanrooms, such as recommending and mandating Good Manufacturing Practices (GMP) to limit the risk of contaminants being transmitted into the drug processing environment.
One of the drug production processes that has a heightened risk of contamination is nearing the final stages of the fill-finish manufacturing process, which is the process of filling vials with drug product, which are, on occasion, lyophilized.
Knowing what the risky and critical areas are during the fill-finish process and determining how to track particle exposure in the appropriate manner is key to the prevention of any contaminants entering a drug product that could present life-threatening health risks to a patient.
This article outlines particle monitoring (viable and non-viable) throughout the fill-finish process of drug manufacturing. It also describes the regulatory guidelines as well as considerations for the location of airborne particle monitors in the equipment design of the processing area.
Recently, there has been an enhanced focus on new biologic therapies and vaccine production, the majority of which are parenterally administered. In a 2018 market report, it was stated that ‘50% of the drugs in the clinical pipeline are made up of biologics’.1
Foreign particulate contamination in parenteral drugs could pose a severe risk to patient health and is still the main reason for drug recalls. Terminal sterilization, where only the final product is exposed to sterilization, is the method of choice for decontaminating injectable products.
Figure 1. Your Complete Vial Journey – From Bulk API to Production. Image Credit: SP Scientific Products
However, not every product, especially biological formulations, can endure the high temperatures needed to accomplish full sterilization. Aseptic processing only facilitates sterilization of each component as part of the manufacturing process and not the final product.
This set-up poses a greater challenge with a greater risk of contamination at various steps in the aseptic filling process and facilitates the aseptic manufacture of products sensitive to terminal sterilization.
Process of fill-finish
The final component when manufacturing aseptic products is the fill-finish operation. At this late stage of production, the biopharmaceutical product is of high value; therefore, any loss of product or contamination would be costly for the manufacturer.
The journey of the vial throughout the fill-finish process, as shown in Figure 1, starts with a wash, and then it is sterilized and depyrogenated prior to being filled with a drug product. Subsequently, a stopper will be added, and, in some instances, the product will be lyophilized.
Finally, the vial filled with product will be capped, washed externally and then loaded onto a tray for evaluation, labeling and packaging.
Accuracy, control and monitoring are key considerations in the fill-finish process.
Environmental monitoring should be able to determine potential contamination routes swiftly so that the necessary course of action can be executed before any extensive contamination in the product occurs.
Tracking the fill-finish process necessitates the complete monitoring of each interactive element of the operation, including interactions between personnel, sterile filtered product, cleanroom and support facilities, the fill-finish equipment system, and sterilized filling components.
The integrity of these elements is compromised by airborne or surface particles in the environment, especially those generated by equipment and human contact, both of which are the prime focus of the particle monitoring process.
On average, a human sheds around ten million particles a day, and these particles are transported by their clothes and shoes when they move through the cleanroom specifically where areas of skin are exposed due to poor aseptic gowning techniques.
Other criteria, such as air velocity, direction, humidity, temperature, and pressure differential, can also impact the way the particles move in the cleanroom setting. For instance, the pressure cascade allows air to flow from the cleanest area, generally the filling area, to the area that is the least clean, so any contaminants will flow away from the filling process.
Contamination throughout the filling, stoppering, and capping processes can be mitigated by placing a simple physical barrier between the filling operation and operators – this could be a restricted access barrier system (RABS) or isolator which limits human contact with the sterile products - or more complex semi-automated or fully automated equipment.
Isolator systems that use glove box technology to reduce operator contact with the drug product are generally biodecontaminated by vaporized hydrogen peroxide. The equipment within a standard isolator is separated into an upper ISO 5 compliant isolator area and a lower technical area divided by a barrier plate.
They are a good substitute for the conventional barrier equipment installation but can be difficult to install, validate and operate; they also need a considerable amount of capital investment.
The RABS is a barrier method that utilizes glove box technology in a traditional aseptic core cleanroom. The benefit of the RABS method is a reduction in initial investment cost and rapid validation. However, RABS can be more expensive to operate and lead to greater aseptic risks compared to isolator systems.
Even when using a fully automated enclosed system, there are periods when impromptu interventions may be necessary, and the classified area is violated for human intervention, so ensuring the potential for contamination is monitored on a regular basis is vital to instill confidence in the sterility of the final product.
The important nature of aseptic manufacturing for parental drugs requires global regulatory requirements. Each country around the world has its own individual regulatory guidance, which is not yet congruent, but they all describe the stages of production that are vulnerable and measure similar parameters regardless of manufacturing scale.
The FDA highlights the importance of maintaining an environment of exceptionally high quality throughout the fill-finish stages of production, where containers are vulnerable state to being exposed to environmental particles.
Good Manufacturing Practice (GMP) guidelines in both the USA (21 CFR parts 201, 211 and 600-680)2 and Europe (EudraLex GMP volume 4)3 state that there is a requirement for prolonged monitoring and constant data collection at each stage of the fill-finish process.
Monitoring must be initially conducted during facility performance qualification (PQ) to categorize the working area, periodically for requalification, and regularly during operation.
Figure 2. Complete Fill Finish Line in an Isolator. Image Credit: SP Scientific Products
The GMP guidelines also draw attention to the fact that the whole process of fill-finish must be conducted in a ‘clean’ environment in accordance with ISO standards (ISO 14644 (4), 21501 (5), 14698 (6), EN17141 (7)).
These standards are able to establish the classification of clean air by the content of particles in the air, and action levels of microbiological quality are advised. ISO 14644 and 21501 standards detail how to properly assess contamination in a cleanroom related to the presence of airborne particles.
These guidelines clearly describe the classification of these particles and the testing methods needed to monitor them and offer guidelines on the development of the cleanroom, which include the performance, location and calibration of particle counters.
Control and evaluation of biocontamination are also described in ISO 14698 (in the EU, this has been replaced by EN17141).
Particles monitored in the fill-finish process are classified by size. In the FDA guidelines, if particles exceed 0.5 microns (µm) at either rest or in operation, they are recorded. This would show bacteria (0.5 – 15 µm) or fungal spore (> 0.8 µm) contamination.
The European guidelines divide this category further into particle sizes of 0.5 – 5 µm and ≥ 5 µm and differentiate between rest (stopped for at least 15 minutes) and in operation. Other components that will affect the quality of contamination detection are affiliated with the sampling protocols.
Establishing how many samples, what the frequencies of the samples are, and the location within the workspace is another crucial decision to be rationalized. Once the areas prone to contamination during the fill-finish process have been established, methods to determine and monitor airborne particles are put in place.
Monitoring of airborne particles
Airborne particles are classified as extraneous contaminants (non-viable, NVP) or viable biological microorganisms according to the regulations for aseptic manufacturing. Viable microorganisms are those that can cause illness to a patient, but NVP can also be dangerous when administered.
There are numerous methods to direct, capture and measure these airborne particles. Most of these function by monitoring the airflow actively or passively via the incubation of agar plates, or a mixture of both.
The method chosen needs to show high efficiency in capturing physical particles, and, in the case of viable particles, the agar plates must preserve their biological ability to grow.
Regulations also outline how the monitoring system should be calibrated and qualified and how to also preserve the sterility of the equipment, e.g., using sterilizable stainless steel or single-use material.
1. Viable particle monitoring
Viable particles can be monitored actively using an air sampler with the ability to capture particles passed onto media (water or gel) from just one cubic meter of air for subsequent incubation over a period of 48 hours. Each batch is then quarantined until the media has been evaluated for colony-forming units (CFUs) growth.
If positive bacterial growth occurs, a formal investigation is necessary. Conversely, passive monitoring uses settling plates of agar that are placed in the open environment and capture particles during a 4-hour period, after which they are incubated and analyzed.
Air samplers vary in relation to how the particles are monitored and by the rate of precision, sampling, and recovery.
Each method has its pros and cons in a laboratory and production setting, and no individual model can overcome all the limitations. This results from active instruments that are generally thought to be quantitative, while those from passive settling plates are considered qualitative or, at best, somewhat quantitative.
An impaction air sampler pulls the air in through small holes in the sampler, and microorganisms are deposited onto an agar plate. Air is drawn in by a vacuum pump, and in the case of the slit-to-agar air sampler (STA), this is via a standardized slit, whereas in a sieve impactor, it is passed through a perforated cover with predetermined sized holes (Figure 3).
Figure 3. Type of Sieve Impactor for Viable Monitoring. Image Credit: SP Scientific Products
These are the two air samplers that are the most popular as they are small and easy to install in any area. Sterility can be preserved in the entire unit by steam sterilization if it is manufactured from stainless steel or using single-use components.
In a closed system, for instance, in an isolator or RABS, sieve impactors are the most advantageous as they can be acquired as self-contained, portable units and systems for complete integration with the aseptic line.
A centrifugal-based monitor is a good alternative to this type of air sampler. This kind of sampler draws air into the head through a rotating vane mechanism (centrifugal propeller sampler). The microorganisms are expelled from the air and onto strips of agar with a centrifugal force.
Although it is flexible and convenient, it can be limited, and there are doubts as to whether it can acquire all viable particle sizes and, therefore, efficiency is reduced.
Another mechanism used to collect airborne viable particles employs a filter to capture any microorganisms from the air, which is then moved to a culture medium and subsequently incubated.
If the filter is fabricated from gelatin, it limits desiccation and damage to the microorganisms that may occur with other types of membranes, although gelatin membranes are typically fragile and not so easy to handle.
Particles can also be captured from the air and fed into a liquid medium. Known as impingers, these air samplers are comprised of a custom tube made from glass or perfluoroalkoxy (PFA).
Air passes through the tube and the particles enter a specified liquid. Aliquots of the liquid are then plated onto agar to identify the microbial content. Colonies form on the medium where the organism impinges.
Most of these methods are dependent on the culture of microorganisms, but these techniques have limitations, the most significant factor being the time needed to culture the microorganisms.
The data is therefore always retrospective and does not facilitate direct corrective action. Another hazard of cultured methods is the dehydration or desiccation of the media, which will kill the microorganisms before they have been able to grow.
In recent years, some advanced technologies have been developed for sampling airborne particles that can identify the numbers in real-time and can detect microorganisms that cannot be recovered using traditional microbiological agar plate methods.
Laser-induced fluorescence (LIF) is a real-time airborne microbial detection technique predicated on the scattering of light. It also has the advantage of being able to identify and distinguish between viable and non-viable particles since microorganisms are comprised of relatively high concentrations of fluorescent molecules and therefore tend to scatter light at various wavelengths.
There is, however, a greater risk of seeing false positives, as not all particles that fluoresce are colony-forming.
2. Non-Viable particle monitoring
Although the detection of viable microorganisms is of significant concern to the production process of drug products and patient safety, non-biological foreign matter, e.g., dust and particulates, can also be detrimental to a patient’s condition.
It has been noted that the presence of non-viable particles can also impact product yield negatively when a cleanroom has been violated after rejection, so it is crucial to monitor these airborne contaminants as well as viable particles.
A light scattering device can be used to determine the size and amount of total viable and non-viable particles and offer direct results in real-time. A beam exits a laser diode and as it comes into contact with the contaminating particle, the laser is scattered.
This scattering is presented on a photodetector which transmits an electronic signal relative to the size and number of the particles (Figure 4). Where the viable particle monitors measure actual microorganisms, non-viable particle monitors measure particles calibrated against latex spheres meaning actual particle sizes could differ by +/- 20%.
Figure 4. Light Scattering Particle Counter – Principle. Image Credit: PMT (GB)
When a particle monitor is being installed, the speed of the airflow and length of tubing need to be taken into account in line with the guidelines for GMP. The ISO 14644-24 standard states the tubing length should be no more than 1 meter with minimal bends to reduce the loss of the larger ≥ 5.0 µm particles.
Particle loss in tubing can also be the result of a lack of turbulent airflow identified by a dimensionless Reynolds number (Re) which is expressed in terms of tube dimension, speed of airflow and fluid properties.
The Re number can anticipate the flow pattern of a fluid. It is advised that for particle monitoring in a fill-finish process, a Re of between 3,000 and 5,500 would offer suitable turbulent flow.
Including an isokinetic port on a scattering device ensures the capture of larger macroparticles (> 5.0 µm), making sure that they are not impeded when being pulled into the inlet. The port also makes sure particles travel at the same speed as the aesthetic environment (0.45 m/ second or 90 ft/minute) which is a requirement of ISO 5.
Location of particle monitoring systems
Knowing precisely where the high-risk areas of particle exposure and monitoring are can be a difficult but key decision as it impacts product sterility and patient safety directly. Poor risk assessments can lead to insufficient monitoring results with badly positioned monitors misrepresenting the status of contamination.
As the users of the aseptic line must explain the location of the particle monitors and justify their decision to the regulatory agencies, it is sensible to consider the space needed for these monitors early in the design of the project.
In aseptic manufacturing of drug products, the key areas with the highest risk of contamination are all within the fill-finish process, but it is also crucial to take into account the dynamics of the cleanroom as possible sources of contamination as well (i.e., air handling systems, overall room size, and equipment location).
Within the cleanroom, an open barrier door system can be the most significant risk of contamination and is not considered best practice, but if an open barrier or RABS system cannot be avoided, it is necessary to carry out additional risk-based qualification control measures.
As isolators are closed throughout the fill-finish process, they present the lowest contamination risk.
Within the isolator/RABS system, zones can be assigned to the filling lines so that focus can be placed on risk assessment, and any area compromised can be isolated and corrected without disrupting other areas.
The critical zones that are most vulnerable to contamination are defined as the areas where vials are exposed to the environment, such as during filling, exiting from the depyrogenation tunnel, loading into the lyophilizer (if required), stoppering, and capping.
When taking the location of the particle monitors into consideration during this process, the ISO 14644 standard (updated in 20154 ) offers a reference table that details the number of locations needed based on room size and is calculated by applying a statistical approach that takes a heterogeneous distribution of particles into consideration at each location (hypergeometric model of particle distribution).
The monitors are placed in areas selected by risk within the critical zones. For the monitoring of viable particles, monitoring should be conducted within 12 in (30 cm) of the point of filling, whereas, for the monitoring of non-viable particles, at least one monitor must be placed within each zone based on risk assessments.
Figure 5. Viable and non-viable particle monitors. Image Credit: SP Scientific Products
If using settle plates, these are usually placed adjacent to the viable/non-viable devices. Figure 6 displays an example of particle monitoring in an aseptic fill-finish process.
Enclosures are divided with mouse holes that have been included to separate the risk zones. An isolator is employed for vial filling and loading (as part of the lyophilization process), which are high-risk processes and incorporate viable, non-viable monitors and settle plates close to each crucial point.
A transfer conveyor carries the vials between these two activities and necessitates only a non-viable monitor as it is not thought to be a critical zone.
A RABS is then employed to transport the vials over to the capping machine. Once the vials have been capped, they are extracted from the RABs and moved to the external vial washer under clean room conditions.
Figure 6. Aseptic Fill-Finish Line Showing Zoning and Locations of Particle Monitors. Image Credit: SP Scientific Products
Since capping is not considered a critical zone, a viable sampling instrument is not necessary.
Supports for settling plates are generally placed next to all non-viable monitors for use throughout the validation and production activities, according to the risk assessment. Eliminating mouse holes between zones can reduce the quantity of non-viable monitors necessary, but any intervention in the zone would then jeopardize a larger number of vials.
Although this example highlights the critical zones most at risk of contamination during the fill-finish process, there are periods when additional monitoring may be necessary. Extra monitoring could be beneficial at points of human entry when loading stoppers or for fault correction.
Also, in areas that contain automated systems, the moving of robots could create an airflow that generates particles increasing the risk of particles entering a vial. Evaluation of these scenarios should be conducted during the risk assessment and validation activities to establish whether particle monitoring is necessary.
When determining a new fill-finish operation or modernizing a facility with new equipment, regulations for aseptic manufacturing can be hard to navigate. It may be beneficial to engage with equipment suppliers early on in the process to source advice on the location of appropriate monitors for environmental contamination.
Most suppliers have comprehensive experience in the development of cleanrooms and locating possible high-risk contamination areas along with the latest knowledge of the suitable regulations.
Conducting a formal risk assessment prior to the transfer and deposition of particles in the specific environment can be crucial when making early location decisions. There are mathematical models that can be accessed to calculate these values and are routinely used as part of a Quality-by-Design (QbD) approach to drug manufacturing.9
There are comprehensive regulations and guidelines from the US FDA, the EU and other national bodies on GMP that describe aseptic processing and offer information to help establish key high-risk areas and outline methods used to protect the drug product from contamination of both viable and non-viable particles.
Knowing the characteristics and limitations of barrier systems and the different types of airborne particle monitors will help when it comes to making the appropriate decisions for a fill-finish facility.
It is projected that the global biopharmaceutical market will grow ≥12%8 over the next few years. Aseptic manufacturing and assisting technologies such as particle monitoring will be a key element in providing safe and effective drugs for the future.
- The Lyophilization Services for Biopharmaceuticals, 2017-2027, Report by Research and Markets, April 2017 https://www.researchandmarkets.com/reports/4229083/lyophilization-services-for-biopharmaceuticals
- US FDA guidance for sterile drug products and GMP (https://www.fda.gov/regulatory-information/search-fda-guidancedocuments/sterile-drug-products-produced-aseptic-processing-current-good-manufacturing-practice)
- EU guidelines for GMP (https://ec.europa.eu/health/documents/eudralex/vol-4_en)
- ISO 14644 cleanroom classification and testing (https://www.iso.org/standard/53394.html)
- SO 21501-4 Particle counter performance and calibration in controlled environments (https://www.iso.org/standard/58073.html)
- ISO 14698 biocontamination control in cleanrooms (https://www.iso.org/standard/25015.html)
- https://www.cleanroomtechnology.com/news/article_page/EN_17141_Understanding_the_risks_of_microbiological_ contamination_in_pharmaceutical_cleanrooms/175973 Summary of EN 17141
- Radar, R.A. and Langer, E.S. Biopharma Manufacturing Markets. Contract Pharma Trends in Biologics Manufacturing. Nov 2018 https://www.contractpharma.com/issues/2018-05-01/view_features/biopharma-manufacturing-markets/
- James L. Drinkwater. The challenges of riskbased environmental monitoring in sterile product filling. European Pharmaceutical Review. February 2016 https://www.europeanpharmaceuticalreview.com/article/39170/the-challenges-of-riskbasedenvironmental-monitoring-in-sterile-product-filling/
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