Validation of a Depyrogenation Tunnel
This blog post will concentrate on the main points of the Process Design phase and the Process Qualification phase.
Introduction
Dry-heat depyrogenation is the primary method for the inactivation of bacterial endotoxin through thermal destruction, and it is commonly used with heat-resistant materials, such as glassware.3,4 The critical process parameters in a dry-heat depyrogenation process are time and temperature.2
The production of parenteral products requires products to be sterile as well as to be free of pyrogens1. Endotoxin is the most significant pyrogen in the health care industry.4 These are high-molecular weight complexes associated with the outer membrane of gram negative bacteria.4 A depyrogenation tunnel is typically used for the inactivation of pyrogens prior to aseptic filling of glass containers, and comes after the washing step. This type of tunnel provides an automated continuous depyrogenation process and usually consists of three zones: a pre-heating zone to pre-warm the glass containers; a hot zone, where the containers are exposed to the required temperature the sufficient time to achieve the depyrogentation effect expected; and a cooling zone, where the containers are returned to room temperature prior to leaving the tunnel and entering the filling environment.3 The exposure time of the load inside the tunnel is a function of the length of the tunnel, temperature and the speed of the conveyor belt. The qualification of a depyrogenation process in a tunnel involves the equipment qualification (installation qualification, or IQ, and the operational qualification, or OQ) and the depyrogenation process qualification (PQ).5
A validation lifecycle approach is recommended to develop a thorough understanding of the equipment and process by investing more time on the process design phase, prior to validation, and then by the continued monitoring of the process after validation to ensure that the process is delivering the expected quality while confirming the state of control of the process.5, 6 This paper utilizes the lifecycle approach and focuses on the first two stages: Process Design and Process Qualification to provide recommendations for a standardized and science-based approach for the qualification of a depyrogenation tunnel.
Lifecycle Approach to a Depyrogentation Process Validation for a Depyrogenation Tunnel
The lifecycle approach to validation is recommended in the 2011 US Food and Drug Administration guidance document, titled “Process Validation: General Principles and Practices,” where it states that “the lifecycle concept links product and process development, qualification of the commercial manufacturing process, and maintenance of the process in a state of control during routine commercial production.”6 According to the lifecycle approach, the validation can be divided into three stages:
- Stage One: Process design
- Stage Two: Process qualification
- Stage Three: Continued process verification
Some of the main deliverables of Stage One include the development of the user requirement specification (URS), the definition of the critical quality attributes (CQAs) and critical process parameters (CPPs), the development of the depyrogenation process and the creation of the standard operating procedure (SOP). During Stage Two, the equipment qualification is completed with the performance of the IQ and OQ and the depyrogenation process is qualified during the performance qualification (PQ) according to the approved protocols. During Stage Three, the process is continuously monitored to confirm the expected results and the state of control of the process.
This concept paper will concentrate on the main points of the Process Design phase and the Process Qualification phase.
Depyrogenation Process Design
During the Process Design phase, the specifications of the depyrogenation tunnel are defined in the URS. Operational specifications include the capacity of the tunnel in containers/hour according to production requirements, specific load size (e.g., volume and dimensions of glassware containers), temperature range and conveyor belt speed.
As for the critical process parameters (CPPs), these are time and temperature for a dry-heat depyrogenation process. The vials are driven by a conveyor belt through each of the three zones. In the hot zone, dry air is heated to the specified temperatures through heat exchangers in order to heat the containers. The exposure time of the load inside the tunnel is a function of the length of the tunnel, the temperature and the speed of the conveyor belt.
The process operational parameters of a depyrogenation tunnel should be designed to achieve at least a three-log reduction of bacterial endotoxin.3,7 The temperature of the hot zone in depyrogenation tunnels is usually set between 220°C and 350°C1 . It is important that all the different vials which are going to be depyrogenated are exposed to at least the defined temperature and for not less than the time determined in the design phase. The European Pharmacopeia Chapter 2.6.8. establishes dry heat at a minimum of 250°C for at least 30 minutes for the depyrogenation of materials such as glassware. For higher temperatures the depyrogenation time required might be only a few minutes.
Similar to the F0 value for steam sterilization, Fh is a measure of heat input and is used to calculate the time in minutes equivalent to a lethality or endotoxin destruction effect delivered by dry heat at 250°C. For depyrogenation the minimum z-value is set at 46,5°C. Although there is no minimum Fh value required for depyrogenation, the determination of the Fh value for each probe may be helpful to ensure consistency and reproducibility of the depyrogenation process.3
Particle count in the depyrogenation tunnel should be appropriate to the exiting environment classification.8 Operational qualification should verify that the HEPA-filtered aseptic environment is maintained within the specifications for an ISO 5/Grade A environment.3 Unidirectional air flow is required to ensure that clean air is always supplied for the heating of the containers. Pressure difference between the zones of the tunnel is needed to avoid air moving from dirty to clean.2 The minimum differential pressure depends on the design of the tunnel and therefore the specification should be checked with the manufacturer.3,8
Figure 1: Scheme of the pressure cascade in a depyrogenation tunnel. The image is courtesy of Syntegon.
Empty and loaded chamber temperature studies can be performed as part of the factory acceptance test (FAT) and then repeated after equipment installation and commissioning at the manufacturing facility, as part of the site acceptance test (SAT) and/or as part of the IQ/OQ. Empty chamber temperature distribution studies are necessary to confirm that the air balance and heated air supply will provide even heating. For these studies thermocouples are placed inside the tunnel equally distributed. The variability in the temperature in the different areas of the tunnel depends on the design and expected variability may therefore be derived from manufacturer´s specifications. Loaded chamber heat distribution studies are also performed to assess the impact of the load in the heat distribution uniformity of the hot zone in the tunnel.3
Heat distribution inside the load can vary depending on the load mass, configuration and other parameters.3 For this reason, heat penetration studies are done to help determine “worst-case” conditions in the load and cold spots. Heat penetration studies confirm the temperature of the load under operating conditions reaches and maintains depyrogenation temperatures. Worst-case operating conditions (e.g., increased belt speed and lower temperature set point) are used for these studies. Heat penetration studies can be done concurrent with loaded temperature distribution studies.3
The “depyrogenation dwell time” is the period during which the containers remain in the hot zone of the tunnel. The dwell time can be derived based on empty and loaded chamber studies.3
After the containers have gone through the hot zone for the duration needed for the depyrogenation, they are cooled down to room temperature in the cool zone prior to the aseptic filling. In order to mitigate any risk of microbial contamination of the containers passing through this zone, it is recommended that the environment is sterilized prior to each filling campaign. This sterilization process is generally by dry-heat and should be validated to reach at least a six-log reduction of bacterial spores. For the validation, dry-heat-resistant bacterial spores should be used as biological indicators, such as B. atrophaeous spores.
Depyrogenation Process Qualification
During Stage Two or Process Qualification phase, the depyrogenation process as designed in the previous phase is qualified. The equipment qualification is completed with the realization of the IQ and OQ and the depyrogenation process is qualified during the PQ according to the approved protocols. During the PQ of a depyrogenation tunnel endotoxin reduction challenge studies are performed and should confirm that the tunnel under production operating conditions is able to achieve a minimum three-log reduction of bacterial endotoxin. These studies typically involve inoculating bacterial endotoxin, e.g. 5000 endotoxin units in the containers to be depyrogenated and verifying after the reduction after exposing the components to the designed conditions with the depyrogenation tunnel. The disposition of the components in the tunnel should be representative to production conditions. An overkill approach can be used and consists of verifying greater than a three-log endotoxin reduction under worst-case conditions, which might include an increased belt speed and lower temperature set point. Endotoxin spiking method and recovery should be designed and determined prior to challenges studies. Recovery studies should be performed with a carrier of the same material as the ones to be depyrogenated in the tunnel.3
Table 1 summarizes the main points during depyrogenation process design and qualification and the potential root causes when failure occurs.
Table 1: Points to consider for the qualification of a depyrogenation tunnel
| Topic | Quality attribute | What to monitor/observe/acceptance criteria | Test Methodology | Potential causes when failure occurs |
|---|---|---|---|---|
| Equipment Construction Verification | Tunnel is built as specified | - Process and instrument diagram walkthrough is performed to confirm that all unit options were built as specified | - Check installation and P&ID is according to GMP design and URS. - Check material of construction certificates | - URS not clear - Factory failures |
| Temperature control and monitoring | - Thermocouples are calibrated. - Temperature is controlled and monitored. | - All thermocouples are calibrated to show accurate temperature at all times during the process. - Temperatures should be recorded. | - Check thermocouples calibration certificates. - Verification of temperature controller and recorder | - Temperature control is not working - Incorrect programming - Equipment component malfunction |
| Conveyor belt speed control and monitoring | - Conveyor belt speed controller is calibrated. - Conveyor belt speed is controlled and monitored. | - Conveyor belt speed control and monitor system works as specified | - Check belt speed controller calibration certificate. - Verification of speed controller and recorder | - Belt speed control is not working - Incorrect programming - Equipment component malfunction |
| Heat distribution with empty chamber (Empty chamber temperature studies) | - The temperature inside the tunnel in different positions with the chamber empty reach the specified temperatures for depyrogenation process. | - Heat distribution in the tunnel with empty chamber is even and is compliant to the specifications defined in the process design - The variability in the temperature in the different areas of the tunnel depends on the design and expected variability may therefore be derived from manufacturer´s specifications | - Temperature mapping with tunnel chamber empty. - Thermocouples are placed inside the tunnel equally distributed. - Temperature variability in the different positions should be checked against manufacturer´s specification. | - Incorrect design - Factory failures |
| Heat distribution with chamber loaded (Load temperature distribution studies) | - The temperature inside the tunnel in different positions with the chamber loaded reach the specified temperatures for depyrogenation process. In some cases, these studies are done concurrent with the heat penetration studies: | - Heat distribution in the tunnel with loaded chamber is even and is compliant to the specifications defined in the process design | - Temperature mapping with tunnel chamber loaded. As for heat penetration studies, thermocouples are placed inside the load and temperature in all spots should meet at least the required temperature for depyrogenation process | - Incorrect design - Factory failures |
| Topic | Quality attribute | What to monitor/observe/acceptance criteria | Test Methodology | Potential causes when failure occurs |
|---|---|---|---|---|
| Air velocity and laminar air flow | The environment in the tunnel complies with a Grade A room/ISO 5 | - Air velocity: Critical Areas should have laminar airflow of 0.45 meters/second (or 90 fpm), with a tolerance of ± 20% around the setpoint8 | - Anemometer might be used to measure air velocity | - Air supply damper not correctly set - Air velocity control system not working - Incorrect programming |
| Particle count/Environment classification | The tunnel meets specifications for total particulates for Grade A/ISO 5 | - Total particle count should verify: - <= 0.5 µm/m3: 3520 particles or <= 0.5µm/ft3: 100 particles (according to EU Annex 1 and ISO 14644) | - Check total particle count and verify according to acceptance criteria - Filter integrity test for the HEPA filters | - Filter damaged - Filter is incorrect installed - Particle control and monitoring is not working |
| Differential pressure | Differential pressure exists between the sections. Air shall not move from dirty to clean | - The minimum differential pressure depends on the design of the tunnel3 and should be checked with the manufacturer. - Air shall not move from dirty to clean. | - Differential pressure should be checked according to manufacturer specifications. - The level of the separators should be considered and documented3 | - Pressure control system is not working - Incorrect programming |
| Sterilization of cold zone | - Cold zone of depyrogenation tunnel should be sterilized prior to aseptic filling to mitigate the risk of microbial contamination to the already depyrogenized vials. | - Supposing sterilization with dry-heat is used, demonstrate at least a 6-log reduction of a dry-heat resistant spores (B. atrophaeus) using dry-heat. | - Use BI strips of B. atrophaeus spores 10^6 hanging from the cool zone of the tunnel, near the position where the load should be. - Run sterilization process and verify total kill of all BIs. | - Load is incorrect - Heat distribution is not working correctly - Air velocity incorrect - Temperature sensors are not working/Temperature control and monitoring system not working |
| Endotoxin reduction challenge studies | Process conditions with the tunnel should achieve the depyrorenation effect expected. | - Demonstrate at least a three-log reduction of bacterial endotoxin. | - Spike load with endotoxins - Run depyrogenation process - Perform endotoxin recovery - Determination of log reduction achieved - Positive and negative controls are needed | - Load is incorrect - Heat distribution is not working correctly - Air velocity incorrect - Temperature sensors are not working/Temperature control and monitoring system not working - Conveyor belt control and monitoring system not working |
Acknowledgement
The author(s) would like to thank members of the ISPE Sterile Product Processing Committee for the opportunity to post this blog and Christian Mrotzek, Klaus Ullherr, and Michael Meyer for original the concept of the blog series covering Critical Quality Attributes essential in aseptic manufacturing of parenteral drug products. Additional thanks to Lukas Munzinger, Michael Meyer, and Alexander Wolf for technical review, comments, and input on the blog.
