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Peer Review Article | Open Access | Published 18th December 2025
Protective Airflow in Aseptic Manufacturing: Ensuring First Air Integrity
Dr. Birte Scharf*, Dr. Hussein Bachir*, Di Morris, James L. Drinkwater | EJPPS | 304 (2025) | https://doi.org/10.37521/ejpps30401
Summary
In GMP‑regulated aseptic filling operations, contamination control relies on complementary strategies including protective airflow and barrier systems. Protective airflow delivers High Efficiency Particulate Air (HEPA)‑filtered air directly to critical processing zones, preventing airborne contaminants from reaching exposed product. Barrier systems physically separate personnel from the process and should only permit controlled transfers and have limited interventions, thereby minimizing surface contamination risks¹.
The practical implementation and interpretation of protective airflow in relation to ‘First Air’ integrity has long been a subject of discussion within the industry. Recognizing this need, the PHSS formed a dedicated focus group with support from the isolator manufacturer Franz Ziel GmbH to research this topic in depth and develop a harmonized understanding of protective airflow principles in aseptic processing environments.
Protective Airflow and First Air Integrity
Central to the protective airflow concept is ‘First Air’, as defined in EU GMP Annex 1: “filtered air that has not been interrupted prior to contacting exposed product and product-contact surfaces, with the potential to add contamination to the air prior to reaching the critical zone”, e.g. open vial pathway, or stopper trackway ². To uphold First Air integrity, the airflow must remain continuously unidirectional from HEPA outlet to critical surfaces. During all critical steps - including routine interventions - First Air must adhere to six essential rules:
Initiation at the HEPA filter (Grade A cleanliness)
Air velocity maintained at 0.45 m/s ± 0.09 m/s (guidance value)
Preservation of unidirectional airflow path integrity
Interaction only with surfaces that are non-particle-shedding and either pre-sterilized or fully bio-decontaminated (validated automated sporicidal decontamination to achieve zero CFU recovery)
fully sporicidal bio-decontaminated surfaces can be accepted for statically installed parts, e.g. air diffusers, velocity probes
fully sporicidal bio-decontaminated surfaces can be accepted for moving parts with special considerations to be not impactful to First Air, e.g. filling needle holders
Allowance for minor, localized turbulence, provided it introduces no particulate or microbial contamination (refer to Figure 4)
Prevention of any re-entrainment of air from lower-grade zones or areas below critical process equipment
First Air is specifically targeted to critical process areas (CPA) like open container pathway, the point of fill, and the stopper pathway: areas where any airborne or surface-derived particle could become intrinsic to the final product¹.
Figure 1 and Figure 2 provide schematic representations of different vial filling lines operated within isolators, illustrating the location of critical process area (shown in red) relative to the surrounding isolator environment (shown in yellow). The area immediately surrounding the CPA must also meet Grade A conditions. Although the same high standards of cleanliness and airflow quality apply, the operational criteria in these Protective Airflow Area (PAA) are somewhat less stringent than those defining the core critical process area.
Typical requirements for the Protective Airflow Area (PAA) include:
• Airflow design ensuring protection of the critical zone from any air reflux, obstruction, or localized turbulence that could compromise First Air-integrity.
• Maintenance of Grade A air cleanliness, even where local air velocities may vary and unidirectionality is not fully uniform, provided no reverse flow or entrainment occurs.
• Use of materials that don’t compromise the requirements of Grade A (low bioburden for non-sterilizable items prior to decontamination) to prevent indirect contamination transfer into the CPA.
These rules aim to ensure that the protective airflow envelope remains intact throughout filling and transfer operations. Initiating directly at the HEPA filter under Grade A conditions guarantees the highest level of particulate and microbial cleanliness. Maintaining the specified air velocity helps balance sufficient momentum to resist disturbances and avoid turbulence.
Air velocity measurement should be performed within the UDAF when the airflow is laminar immediately after the HEPA filter, the so called LF_UDAF zone (refer to Figure 3) above working positions³. Measurement at working positions where high-risk operations are performed is aligned with the Annex 1 requirement, the literature shows that a measurement at working height with complex process equipment in place does not offer advantages⁴. The measurement height will also be discussed in a future PHSS publication. A strictly unidirectional path prevents ingress of lower-grade air, and limiting interactions to non-shedding, sterilized surfaces remove potential contamination sources. Finally, allowing only minor turbulence and prohibiting re-entrainment of degraded air preserve the stability of the First Air shield around open containers and critical process interfaces⁵,⁶.
Given the complexity of aseptic processing equipment within isolators and restricted access barrier systems (RABS), some degree of turbulence is to be expected⁶. Airflow may encounter obstacles and equipment surfaces that cause localized changes in direction or minor turbulence, and the key is to determine whether such turbulence compromises the protective airflow.
Acceptable turbulence is defined by specific conditions that ensure the integrity of the First Air protection. Minor turbulence is acceptable if it is localized and controlled and remains confined within the First Air zone, even when interacting with surfaces such as diffuser screens, velocity probes or process equipment (refer to Figure 4).
In these situations, even after minor disturbances, the airflow must continue on a unidirectional path without drawing in lower-quality surrounding air and the surfaces along its path must not shed particles that could compromise Grade A conditions. In addition, all surfaces contacted by First Air should have undergone effective bio-decontamination and be none-particle shedding.
Conversely, unacceptable turbulence is classified as a deviation because it poses a significant risk of contamination by compromising First Air integrity. If turbulence results in the recirculation of lower quality air that potentially does not meet Grade A standards, the integrity of the First Air protection is compromised. Similarly, turbulence that disrupts directional flow or creates reverse airflow patterns increases the potential for airborne contamination transfer and requires corrective action to restore proper aseptic conditions (refer to Figure 4).
Protective Airflows: UDAF and L‑UDAF
The Pharmaceutical & Healthcare Sciences Society (PHSS) defines protective airflow in two principal configurations: Unidirectional Airflow (UDAF) and Localized UDAF (L‑UDAF)⁷. UDAF generates a uniform airflow from the HEPA filter across the entire Grade A clean zone, ensuring a continuous protection of First Air over filling lines, stopper bowls, and other critical process areas. In contrast, L‑UDAF delivers targeted airflows at specific risk interfaces - such as autoclave unload, mouseholes between CPA, and RABS barrier doors - where a full enclosure is impractical. At Grade A CPA zone interfaces, L‑UDAF maintains local Grade A conditions while allowing excess air to overflow into adjacent zones, thereby preventing ingress of lower-grade air during component transfers and occasional interventions.
Introducing ‘Second Air - Monitoring’
The recent publication⁵ offers a new understanding how First Air protects open vials by creating a shield around them (Figure 5).
This results in a new concept for environmental monitoring in First Air protection zones, based on ‘Second Air monitoring’. ‘Second Air monitoring’ refers to the monitoring of the air formed within the UDAF after it has passed the point of protection as ‘First Air’ (refer to Figure 3). ‘Second Air monitoring’ applies for any part of the open vial and stopper trackway. The understanding of ‘Second Air monitoring’ has consequences for the placement of environmental monitoring positions and includes a link to environmental monitoring risk assessments that will be elaborated and explained within an upcoming article.
Equipment Design and Regulatory Implications
The 2022 revision of EU GMP Annex 1 elevates the importance of First Air to a central design criterion, driving substantial evolution in aseptic filling machinery and robotic systems. Process equipment must follow Quality by Design approach (QbD) and take First Air criteria into consideration.
Special attention is also required in terms of the choice of materials and the technology construction process. For example, for Isolators, all surfaces within the protective airflow pathway, including HEPA air diffuser assemblies, feeder bowls (following sterilization) and defined material transfer interfaces, must be bio-decontaminated using an effective sporicidal agent, such as vaporized hydrogen peroxide. Selecting materials carefully (e.g. GMP grade stainless steel and non-shedding polymers) reduces the risk of particle generation during routine operations. The quality risk management principles embedded in Annex 1 highlights the connection between process monitoring (e.g. air velocities and pressure differentials, as contamination control measures) and environmental monitoring (e.g. particle counts and microbial sampling, that monitor the outcome of control). Annex 1 now explicitly links environmental and process monitoring, requiring real-time alarms and deviation management to detect any loss of First Air integrity at critical points. By incorporating these principles, which have been validated through airflow visualization, into the QbD framework, manufacturers can achieve robust and predictable protective airflow performance that remains resilient under varied operational conditions⁵.
Airflow visualization: Smoke Studies and Computational Fluid Dynamics (CFD)
Smoke visualization studies, as mandated by the latest revision of EU & PIC/S Annex 1, remain the foundational qualification tool for confirming uninterrupted First Air delivery. Designed according to Quality Risk Management principles, these studies employ standardized smoke generators, strategically positioned cameras, and predefined acceptance criteria to demonstrate continuous unidirectional airflow patterns enveloping critical points such as open containers, feeder bowls, point of fill and material transfer ports. Initially, under static, or “at rest,” conditions, smoke is introduced without equipment or product movement to confirm that First Air maintains its integrity even when encountering complex geometric challenges of the integrated barrier and process equipment. Then the system is evaluated under operational conditions (“in operation”), which include setup processes, filling operations, and inherent and corrective interventions, smoke studies capture dynamic interactions and visually document any turbulence or re-entrainment of low-quality air into the clean airflow⁷. Although inherently qualitative, these studies are essential for regulatory approval because they provide immediate, intuitive confirmation that protective airflow patterns align with stringent quality criteria.
“Where unidirectional airflow is required, visualization studies should be performed to determine compliance”². However, while smoke studies provide valuable visual evidence, they may not fully capture the dynamic nature of airflow during actual process operation, as it is not possible to see through the smoke. In such cases, computational fluid dynamics (CFD) analysis offers a particularly advantageous approach. CFD models can simulate a range of operational scenarios, including the effects of moving materials and complex interactions with equipment. This dynamic simulation provides a deeper understanding of airflow speeds, directions and localized turbulence, enabling risk-based assessment that predicts whether observed changes are within acceptable limits. In addition, CFD analysis has the ability to identify potential re-entrainment zones that may not be apparent in a smoke study and therefore serves to complement airflow visualization qualification of airflow patterns by revealing airflow behavior within areas that cannot be visualized alone by smoke studies e.g. within sterilizing tunnels or even inside vials/ product containers.
Visual confirmation of these predicted airflow dynamics through smoke studies remains a key component of qualification, providing assurance that airflow behavior aligns with expectations. Meanwhile, CFD modeling delivers deeper insight into areas that are not easily observed visually, particularly within equipment geometry and contained spaces.
Complementing these two visualization methods, the Limitation of Risk (LR) Method offers a third, more quantitative approach. By integrating a controlled particle challenge - using aerosolized tracers or smoke - with real-time particle counting, the LR Method maps particle transfer from potential release points (for example, glove holes or process equipment surfaces) to points of risk. This enables precise determination of particle migration paths, supporting environmental monitoring location decisions and verifying containment in open pathways such as mouseholes⁸. Because it quantifies particle concentration rather than relying solely on visual patterns, the LR Method provides additional assurance that First Air remains uncompromised even when airflow patterns become complex or when hidden recirculation zones develop.
The LR Method therefore further strengthens this validation by quantitatively confirming particle containment and transfer risks.
Together, smoke studies, CFD analysis, and the LR Method provide a comprehensive validation framework - ensuring that airflow conditions meet regulatory standards and minimize airborne contamination risks during aseptic processing and associated bioburden control process steps.
CFD Protective Airflow visualization at critical process points in Filling operations
The aim of this chapter is to go into the review of different process critical areas, show the CFD visualization of the airflow and extract learnings from these particular images.
Protective airflows at Sterilizing tunnel Vial infeed:
The vial infeed area, specifically at the sterilizing tunnel exit and the turntable transfer point, is a zone of particular interest with regard to airflow management. This is due to the presence of a pressure differential between the isolator chamber (Grade A filling zone) and the sterilizing tunnel. It is imperative that the protective airflows in this area are carefully controlled and monitored to ensure that aseptic conditions are maintained throughout the transition from sterilization to filling.
There are three different conditions that need to be considered in terms of airflow.
Table 1: Different states of the sterilization tunnel door in the aseptic filling process.
Phase | Tunnel door | Vials | Airflow |
Initial phase | closed | No vials | Airflow above the turntable is stable vertical UDAF |
Start-up phase | open | Vials in tunnel | Pressure differential between Grade A filling zone and tunnel creates a cascade effect, driving a protective horizontal airflow through the tunnel |
Production | open | Vials in tunnel, interface and on turntable | Airflow protection is continuously maintained by a combination of downflow and horizontal flow patterns |
In each scenario, the aim is to maintain continuous, high velocity UDAF from the Grade A zone, which is carefully evaluated through CFD modelling.
Scenario 1: Initial phase
During the initial phase, before the vials leave the sterilization tunnel and are transferred to the turntable, the sterilizer door remains closed. Under these conditions, the airflow patterns over the turntable are stable. CFD plot shows that the protective airflows descend smoothly over the turntable surface, with airflow vectors indicating reduced velocity near the flat surface of the table (Figure 6 A) – creating a similar protective shield above the table as for a single vial (see below). When the air approaches the edges of the table and the central opening, it changes its direction from downflow to horizontal. The velocity increases significantly as the air is directed from the complex equipment towards the barrier's return air ducts (Figure 6 B).
Learning: Protective shield is important, should not be disrupted.
Scenario 2: Start-up phase
When production starts and the tunnel door is opened in preparation for vial ingress, the airflow dynamics change. The pressure differential between the Grade A filling zone and the tunnel creates a cascade effect, driving a horizontal airflow through the tunnel. Due to the horizontal air movement as a result of the overpressure interacting with the downward flow from the isolator and the tunnel respectively, there is no supplementary protective shield above the containers as the horizontal airflow is the sole airflow protection. On route to the tunnel the UDAF air passes surfaces inside the barrier e.g. isolator, like the wall and the tunnel door, and can afterwards exchange air within the containers. Therefore, the air pathways contacting surfaces should be non-particle-shedding and fully bio-decontaminated according to the First Air rules. In addition, surface sampling at the end of the batch is considered necessary to monitor Grade A surface conditions have been maintained through operations.
The change from downflow to horizontal flow with high velocity also introduces localized turbulence. There is a notable risk of airflow from below the turntable, potentially drawing contaminants from the baseplate area into the tunnel. It is critical that the air touching the vials does not mix with turbulent patterns from below, which could compromise sterility.
Learning: End-of-batch surface sampling should include the tunnel door and the wall above it (these must be within reach of a glove/tool for swabbing).
Scenario 3: Production
Once the vials are actively exiting the sterilization tunnel, crossing the interface and entering the turntable for filling, the vials on the turntable are protected by the protective shield formed by the UDAF. Overall, the airflow protection is continuously maintained by a combination of downflow and horizontal flow patterns. A particular challenge during this phase is the interaction between the high velocity airflows and the open sterile vials. Inside the tunnel, CFD data shows that the air flows over the vials and can create internal air movement within the vials themselves. The vials are no longer 'dead legs' like in downflow UDAF, they exchange air with the surrounding airflow. This becomes a high-risk zone as turbulence could theoretically introduce airborne contamination into the open vials if the Grade A air protective air is compromised. UDAF from below the turntable, potentially drawing contaminants from the baseplate area, should not interact with or compromise the First Air protective zone above the containers.
Learning: In vertical airflow the protective shield extends over a group of vials, not only a single container. The air movement within the tunnel resulting from the overpressure from the isolator exchanges with air inside the open containers. Air beneath should not interact with the air above.
Airflow at the Point of fill
CFD Case Insight: Shielding the Point of Fill (PoF)
Caveat: The following studies apply to vertical downflow as primary First Air protection, if horizontal airflow is considered to be the primary First Air protection, then further studies will be required to mitigate airborne contamination control to an acceptable level.
CFD analysis at the PoF reveals that vertical downflow UDAF does not ‘sweep’ into open vials but rather forms a surrounding shield of clean air (refer to Figure 5). Inside each container, air velocity approaches zero, effectively creating a static ‘dead air’ zone. As long as the protective shield remains unbroken or uncompromised, airborne contaminants from the surrounding environment cannot penetrate this zone or container. However, when the pattern shifts to horizontal or cascaded airflow, turbulence increases, disrupting the protective envelope and exposing both individual and grouped containers to airborne contamination⁵. These insights underpin both smoke study acceptance criteria and the aerodynamic design of filling machines, emphasizing the need for airflows that are non-compromising First Air integrity over PoFs.
As described in an opinion paper⁵, protective airflow design considerations apply to both single vials and to pre-sterilized (RTU) containers arranged in nests at the point of fill.
For open vials, CFD analysis indicates that the container geometry creates a ‘dead leg’, which reduces the continuous flushing effect of the protective airflow inside the container. Although the directed flow acts as a First Air shield at the filling point, the product itself is not fully suppressed by the airflow and aerosolization can contaminate the outer surfaces of the vial with product⁹. It should also be noted that under these ‘dead leg’ conditions there is no significant evaporation of residual hydrogen peroxide from within the vials if RTU packaged pre-sterilized containers are exposed to VHP/vH2O2 bio-decontamination in material transfer to Grade A.
In the case of pre-sterilized containers arranged in nests, a distinct ‘dead zone’ can form under the tub. This zone can act as a tunnel, facilitating contamination transfer and particle deposition. Therefore, proper airflow design is critical for maintaining aseptic conditions.
In addition to the previous findings regarding the protective shield generated by First Air over a single vial at filling, in these studies the impact of filling needle holders (design and format) on First Air protection was further evaluated via CFD analysis.
Point of Fill with multiple filling needles – impact of needle holder design
Two simulations of the filling needle holder (see Figure 9) reveal that even small changes to the design can significantly impact the protective airflows at the PoF. A filling needle holder with a rectangular shape and no aerodynamic design positioned directly above open vials is a worst-case scenario. Filling machine manufacturers need to take this into consideration during the design phase in order to optimize the design of the filling needle holder for maintaining of First Air protection not only during filling, but also for aseptic setup and intervention manipulations.
Excursus: Robotic design and movements
While robotic automation is valuable for removing human operators from the aseptic core, it is crucial to recognize that robotics alone do not eliminate contamination risks. In fact, the very presence of robotic arms within a Grade A environment can disrupt First Air unless their design and movements are carefully engineered. As emphasized in Annex 1, First Air that passes over any obstacle - whether it be a filling needle holder or a robotic gripper – must continue in a unidirectional flow toward the exhaust pathway. If a robot’s geometry or motion generates turbulence, it can compromise First air integrity and the protective shield that protects the PoF, allowing quality-impacting particles or microorganisms to invade the critical zone, should they be present.
Accordingly, any robotic system used for aseptic filling must follow a QbD philosophy that explicitly incorporates First Air principles. For example, handling tools mounted on robotic arms should be aerodynamically profiled to minimize eddies, and motion sequences should be programmed so that the robot follows good aseptic technique. Just as filling machine designers need to optimize needle holders to mitigate First Air compromise within the CPA (Critical Process Area), robotics engineers must follow the same requirements plus ensure that each joint, linkage, or gripper does not provide or create contamination risks and there are no flow shadows beneath or behind the robot that could provide a wider impact to protective airflows within the PAA (Protective Airflow Area).
Riboflavin cleaning studies have shown that residual contamination often accumulates in robot joints and fixings, underscoring the need for dedicated cleaning protocols and ‘hygienic design’ features (such as appropriate surface finish, minimal crevices) that facilitate thorough sporicidal disinfection¹⁰.
Finally, the assumption - prevalent in some early implementations - that robotics eliminate the need for microbial monitoring in Grade A zones is not correct. Still full qualification and monitoring of the environment per Annex 1, including particle and microbial monitoring, is necessary. Detailed analysis of airflow at robots will follow within an upcoming article.
Protective Airflow at the Stopper Slide and Stopper Bowl
Understanding and demonstrating protective airflow around the complex and critical equipment for the container closure feed system (stopper chute, hopper and stopper bowl) is crucial to maintaining product sterility. This section presents a CFD-based assessment of the airflow around the stopper feed chute, hopper, and stopper bowl under the dynamic conditions of Rapid Transfer Port (RTP) door opening, glove manipulation, and stopper loading. The results show that localized turbulence, if properly managed, does not necessarily compromise First Air integrity. However, a barrier glove intervention can present an increased risk to compromise First Air integrity and special considerations are required. Principally, airflow patterns could remain protective if they follow the rules of First Air: localized turbulence without drawing lower-quality air into the critical zone, no additional particle shedding, and no introduction of microbial contamination. These airflow studies focus on the established design of stopper chute and bowls; other designs may be applicable for stopper feeding that avoid setup pre-VHP.
Stoppers are fed via a RTP port, through a chute, into a hopper and subsequently into a stopper bowl, all parts of the stopper trackway are considered as indirect product contact parts and are therefore sterilized prior to line setup. Three operational scenarios illustrate the protective airflow requirements:
Table 2: Overview of the Stopper Loading Configuration
No | Status | RTP door | Process |
1 | Preparation for production | Closed after VHP/ vH2O2 Bio-decontamination | After setup and vH2O2 gassing cycle, before opening the RTP door for stopper loading |
2 | Production | Open | Stopper loading and normal filling operations |
3 | Preparation for stopper infeed (RTP opened from inside) | Closed à Open | Glove hand entry for RTP door opening (during reloading of stoppers in ongoing filling operations) |
These scenarios consider both static and dynamic conditions, recognizing that real-world aseptic operations involve frequent interventions. The overall aim is to maintain a continuous, unidirectional airflow and to ensure that any airflow pattern changes around the stopper slide and bowl maintain First Air protection.
Scenario 1: Preparation for production
During the initial phase, the RTP door is closed and airflow vectors are predominantly downwards (green vectors), providing Grade A cleanliness control over the feed chute and stopper bowl. The air forms a similar protective shield above the stopper and hopper bowl as for the turntable or open vial. However, the geometry of the equipment, such as the ‘dead-leg’ area within the hopper bowl, causes reduced velocities (blue vectors) and some localized turbulence (see Figure 10). These turbulences, if contained and rejoining the primary downward airflow without entraining air from lower class areas, are generally acceptable and do not constitute a deviation. Complex equipment shapes (e.g., underside of the bowl) inevitably create turbulence. Not all turbulence is a failure or deviation; the focus is on whether the airflow path is interrupted or reintroduced from lower class, higher risk areas below the critical process point where some non-entrained particulates inherently settle. In these studies, the airflow patterns remained consistent with the protective UDAF and there was no evidence of re-entrainment of potentially compromising airflow patterns to Grade A conditions.
Learning: Protective shield above stopper/hoppers - First Air shield is still in place. Turbulence exists under the bowls.
Scenario 2: Stopper infeed (RTP door open)
With the RTP door fully open for stopper loading, the door is not directly above the chute, but remains close to the critical process point. The airflow for this setup for the chute, hopper and stopper bowl in this configuration is similar to the Scenario 1 (Figure 10) except for the door (Figure 11). Figure 11 shows the unidirectional airflow over the door area. Air flows over the open door recombine with the main UDAF path and remain clear of the critical feed chute and stopper bowl area. This localized airflow does not interfere with the protective downflow of the hopper. However, consideration is required through the RTP door movements at stopper bowl re-filling as air that passes over the RTP door would then enter the feeder bowl.
Learning: Air flows over the open RTP door, combining with the main UDAF path maintaining First Air integrity while remaining clear of the critical feed chute and stopper bowl area.
Scenario 3: Preparation for stopper infeed (RTP door opened via a glove from the inside)
Figure 12 illustrates the airflow dynamics during the opening of the RTP door with a glove, highlighting its impact on First air integrity and potentially aseptic integrity. The protective airflow forms around the aerodynamic shape of the glove and recombines into a protective UDAF. However, as the air passes over the barrier gloves, it poses an airborne contamination transfer risk, potentially carrying contaminants into critical areas such as inside hopper and stopper bowl and over container closures. The airflow trajectories show that air passing over the gloves then flows over the sterilized chute and into the sterilized hopper, both of which are indirect product contact surfaces. Given this risk, the integrity of the gloves and their bio-decontamination status (Grade A surface conditions = no CFU recovery) are crucial. Gloves used to open and close the RTP door play a vital role in maintaining aseptic conditions and must therefore be considered critical process gloves to minimise contamination the risk of contamination. It is a given that no barrier glove should contact directly direct or in-direct product contact surfaces but airborne contamination transfer risks are inherent if gloves pass over critical surfaces. The safest approach would certainly be to use RTPs that can be opened/ closed from the outside for isolators, as these do not require such glove interventions. These work best in combination with beta bags that include sleeves and/or collars to bridge the ring of concern and via tilting mechanisms control and direct the stoppers straight onto the sterilised chute and onward to the feeder bowls/ tracks.
As the RTP port door becomes part of the airflow path during stopper loading (Figure 12D), it is fully exposed to the VHP/vH2O2 sporicidal bio-decontamination cycle of an Isolator (via dummy containers to permit door opening) and it is recommended that the RTP door surface is included in end-of-batch surface environmental monitoring. While the chute, hopper, and stopper bowl are standard indirect product contact surfaces, the RTP door can also be an area where contamination might accumulate and transfer as airborne contamination if not properly managed.
Excursus: Glove management and contamination control
In addition to quality by design of the process and equipment (i.e. the design of the RTP port allows for opening/ closing from outside) only with all of the required control measures, proper management of barrier gloves is it possible to minimise contamination risks to an acceptable level. Wherever possible, the glove used at the RTP door should be dedicated to stopper loading operations to reduce the risk of particle or microbial transfer from other operations. If the glove is used beyond RTP door operations, procedural controls must ensure that it remains aseptic as its outer surface consistently needs to meet Grade A bioburden requirements (no CFU recovery). Care must also be taken to avoid any contact between the glove and non-product contacting surfaces including machine baseplates, as even minimal contact can increase contamination transfer risks. Routine integrity testing combined with regular end of batch surface environmental monitoring (using methods such as contact plates or finger swabs), is essential to detect glove integrity compromises that could allow microbes to penetrate and transfer into the Grade A environment¹¹.
Learning: The barrier glove poses a significant contamination transfer risk above indirect product contact parts. Following QRM, ports that are opened/ closed from the outside should be considered.
Conclusion
Protective airflow is essential for aseptic manufacturing as an airborne contamination control measure, with First Air acting as a protective barrier to airborne contamination transfer between the aseptic processing Grade A environment and the sterile product processed. As defined in Annex 1, the First Air concept should be considered as one of the design priorities and performance criterion, verified through airflow visualization. Facility, process and equipment design and associated environmental control are the focus following QRM priorities as ‘Monitoring and testing alone does not provide assurance of sterility’ : Annex 1 clause 2.2
These studies improve knowledge about characteristics of protective airflow including First Air and associated risks to mitigate by design and well-designed procedural control. In this article the studies have a key focus on filling operations and process interfaces (e.g. sterilizing tunnel exit, point of fill and stopper feed system). While local turbulence may be acceptable, under certain circumstances, any reintroduction into First Air protection from areas below the critical process point or from surrounding environments that may include contamination risks, or horizontal disturbances at open containers can compromise protection. These findings emphasize the importance of optimizing equipment or process design (e.g. for filling needle holders or RTP door operations) to maintain First Air integrity.
Looking to the future, future work will elaborate on the concept of Second Air monitoring as an advanced tool for assessing post-First Air environments and linking airflow performance directly with environmental monitoring data. Further studies will address the correlation between measurement position and airflow behavior, establishing criteria for probe height and placement within LF_UDAF zones. Additionally, an upcoming paper will focus on robotic airflow interactions and their qualification within Annex 1 compliance frameworks.
Maintaining First Air integrity ultimately demands a fully integrated approach, combining equipment design, CFD analysis, airflow visualization and real-time monitoring. Only by embedding these principles into Quality by Design and process monitoring strategies can aseptic manufacturers ensure the sterile product is robustly and reproducibly protected in a manner that complies with regulations.
Abbreviations
Abbreviation | Full Term | Short Explanation |
AAS | Grade A Air Supply | HEPA filtered protective Air supply particle concentration qualified but not continuous monitored (for protection of fully stoppered vials) at capping operations |
CFD | Computational Fluid Dynamics | Numerical simulation method for analyzing flow patterns; velocity and flow vectors |
CFU | Colony Forming Units | Measure of viable microorganisms. |
CPA | Critical Process Area | Area directly impacting product sterility (e.g., open containers, point of fill). |
GMP | Good Manufacturing Practice | Regulatory framework. |
HEPA filter | High Efficiency Particulate Air Filter | High-efficiency filter providing clean air. |
LF_UDAF | Laminar Flow Unidirectional Airflow Zone | Zone of laminar flow directly under the HEPA filter within UDAF, velocity measurement applied. |
L-UDAF | Localized Unidirectional Airflow | Targeted UDAF at specific process interfaces that do-not possess a full barrier surround. |
LR | Limitation of Risk Method | Quantitative particle challenge method to map particle transfer paths. |
PAA | Protective Airflow Area | Surrounding Grade A area outside the CPA, still Grade A with protection with less stringent requirements to First Air. |
PoF | Point of Fill | Location where product is filled. |
PrM | Process Monitoring | Monitoring of process parameters such as fan speed, airflow velocity, differential pressures and temperatures, relative humidity if process critical. |
QbD | Quality by Design | Design philosophy embedding quality criteria into equipment/process design. Although the QbD Design space criteria may still apply the principle of designing in quality to maintain contamination control to assure product quality is a further enhancement. |
QRM | Quality Risk Management | Systematic assessment and control of risks per Annex 1. |
RABS | Restricted Access Barrier System | Barrier technology that combines a physical barrier and aerodynamic protection that restricts operator and material transfer access, hence reducing contamination risk. |
RTP | Rapid Transfer Port | Port for closed aseptic transfer of materials. |
RTU | Ready-to-Use | Pre-sterilized, pre-packaged product container and closure components (e.g., vials in nests, stoppers in RTP bags). |
UDAF | Unidirectional Airflow | Uniform, directed airflow pathway. |
vH₂O₂/ VHP | Vaporized Hydrogen Peroxide | Sporicidal agent used for automated bio-decontamination. |
References
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Acknowledgements
The authors would like to acknowledge Alan Moon, Director at AM GMP Limited, for his valuable input and discussions regarding the design of experiments for the filling needle holder investigation.
Special thanks go to Jens Robert, Mechanical Engineer at Franz Ziel GmbH, for performing the computational fluid dynamics (CFD) analyses.
The authors also wish to thank Rick Friedman, Deputy Director of the Office of Manufacturing Quality at the U.S. FDA, for his insightful contributions to discussions on experimental design.
Finally, sincere appreciation is extended to Olaf Ziel and Franz Ziel GmbH for sponsoring this study.
Authors
Dr. Birte Scharf* - Franz Ziel GmbH, Dr. Hussein Bachir* - Franz Ziel GmbH, Di Morris - PNR Pharma, James L. Drinkwater - Franz Ziel GmbH
*These authors contributed equally to this work
* Corresponding author:
Dr. Birte Scharf birte.scharf@ziel-gmbh.com