In terms of sterility assurance, aseptic processing is a more hazardous pharmaceutical process to conduct compared with terminally sterilised products since should microbial contamination occur during dispensing the product, due to its inherent nature, cannot be subjected to a post-filling sterilization process. With aseptic filling the product is sterilized separately, then filled and packaged using sterilized containers and closures in critical processing zones. Several environmental controls, particularly in relation to cleanroom and clean air design, are built into the process to protect the product. Personnel are required to follow strict discipline in relation to good aseptic techniques. One of the periodic assessments undertaken to measure the likelihood of non-sterility is the Aseptic Process Simulation (APS) test (or the ‘media simulation trial’).
By Tim Sandle
With media simulation trials, a microbiological growth medium is used in place of the product and filled as if it were a product under the ordinarily processed conditions. Media fills start at the beginning of filling operations (immediately after the line setup), during and after manipulations and interventions, and until the last vial has been filled. For conducting media simulation trials, regulatory guidance provides an outline of what needs to take place and the acceptance criteria (ideally zero growth in any filled container) that need to be adopted.
The advantage of the media simulation trial over the sterility test is that all media-filled containers can be incubated. Following incubation they can be 100% visually inspected and checked for microbial growth. By this complete inspection, the contamination rate associated with a media fill can be assessed directly.
Media simulation studies are designed to simulate the entire process to evaluate the sterility and confidence of the process. Process simulation studies include formulation (compounding), filtration and filling with suitable media. Simulations are made to ensure that the regular process for product batches repeatedly and reliably produces the finished product of the required quality. To be of value, media trials must be representative of the types of products filled on an aseptic processing line. To ensure that media fills are representative, the fill must replicate the conditions under which product filling takes place, and they must be undertaken under "worst-case" conditions so as to provide a realistic challenge.
This article looks at the regulatory requirements for the APS, some of the design requirements and the importance of selecting an appropriate culture medium.
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GMP logo (designed by Tim Sandle)
Regulations
The U.S. Food and Drug Administration states that the purpose of an APS is to qualify the aseptic process using a microbiological growth medium manipulated and exposed to the operators, equipment, surfaces, and environmental conditions similar to the way the product itself is exposed (FDA, 2004). In addition, EU GMP Annex 1 requires an APS to be capable of:
• demonstrating the capability of the aseptic process to produce sterile drug products
• qualifying aseptic processing personnel
• reflecting typical aseptic operations
The core regulatory expectations are:
1. For three initial qualifying (or repeat qualifying) media fills to be run per line.
2. For media fills to be executed twice per year per line.
3. For personnel to be qualified once per year through a media fill.
4. For media fills to typically be 5,000 or 10,000 units.
5. For media fills to be of sufficient length so that all inherent and corrective interventions can be performed.
6. For media fills not to incorporate poor practices.
7. For the acceptance criteria to be zero.
Therefore, APS studies should simulate aseptic manufacturing process operations as closely as possible, incorporating a worst-case approach. The potential risks to be included are [1]:
• personnel
• equipment
• components
• facility and utilities
The main requirements for performing aseptic process simulations have become more demanding during the last decade; at the same time, the technologies used for aseptic compounding and filling have improved, providing greater protection (including simple-use items and barrier systems, including isolators).
Key considerations
The APS program should incorporate the contamination risk factors to allow an assessment of the state of process control to be made. In doing so, media fill studies should simulate aseptic manufacturing process operations as closely as possible, incorporating a worst-case approach. These include [2]:
Equipment
• Factors associated with the longest permitted run on the processing line
• Number and type of normal interventions, e.g., maintenance, stoppages, and equipment adjustments
• Line speed and configuration
• Lyophilization where applicable
• Aseptic assembly of equipment (e.g., startup)
Personnel
• Number of personnel and their activities
• Shift changes, breaks, and gown changes
• Operator fatigue
Cleanroom operator. Image by Tim Sandle
Operations
• Number of aseptic additions
• Number and type of aseptic equipment disconnect and connections
• Aseptic sample of collection
• Manual weight checks
• Container closure system
• Specific provisions for aseptic processing-related standard operating procedures
Media fill documentation should include the identification of the risk variables in defining the worst case. A protocol in the form of a batch record must be prepared for each run on each line.
Design of a media fill program should include, but is not limited to, the following:
• Identification of the process such as lyophilization, aseptic solid, powder and liquid fill,
• Identification of the room,
• Identification of the filling line and equipment,
• Number of personnel to participate,
• Identification of which personnel teams are to be included. Where filling runs normally include a shift change, then a shift change should be practised during the media filling run,
• Type of container/closure to be used – should compare with routine production,
• Line speed (to be equivalent to production operations, especially with the time that vials spend at the point of filling),
• Number of units to be filled,
• Number and type of interventions,
• Type of culture media to be used,
• Volume of medium to be filled into the containers (so there is sufficient headspace to permit microbial growth),
• Aseptic set up and assembly of sterile equipment,
• Incubator identification and incubation time and temperature,
• Environmental monitoring requirements (these should not be less stringent than those used in a product fill),
• Copy of the batch record to be used,
• Acceptance criteria for the test,
• Description of documentation record for the final report,
• Box or tray number of positive units,
• Growth support testing requirements and results,
• Rationale for worst-case ‘‘parameters’’ chosen (see below),
• Summation of the data from the batch record environmental monitoring samples based upon this information, a conclusion is formulated regarding the acceptability of the manufacturing process and the facility.
The environmental monitoring performed during the media fill should be equivalent to the monitoring performed during a commercial product fill [3].
Matrix approach to design
When several different vial combinations, fill volumes, line speeds and so on are established for an array of different product fill combinations it is acceptable to design a matrix so that worst combinations can be selected and incorporated into the media fill program. The term "worst-case," in the context of media fills, is taken to be the combination of events and circumstances that could, in theory, expose a product (or, in this case, a product simulation) to the greatest chance of microbial contamination [4].
The factors to consider include vials that have the widest neck diameter (thereby providing a larger target for the deposition of contamination), fill speeds or fill volumes (that lead to vials remaining at the point of fill for the longest time), larger media fill runs (where the longest fills, that over time, could lead to a higher frequency of contamination events occurring), and the type and nature of personnel interventions (where the act of a person intervening into a critical zone poses a potentially high contamination risk; some interventions, due to their complexity or time taken to complete, pose a greater risk than others).
No single risk factor necessarily presents a "worst-case." However, one combination of factors will probably present a greater potential risk to a product than another combination and thus may be considered a greater risk and "worst-case". It is the argument of this paper that, by placing these factors into some form of matrix, the "worst-case" is easier to visualize and justify. Once the "worst-case" combinations have been found, it is possible to draw up a rationale for the selection of these conditions for the execution of scheduled media fills.
Culture media (designed by Tim Sandle)
The importance of culture media selection
An APS is usually conducted with a soybean casein digest medium. This can be either a base that is used to make a broth and which is then sterile filtered or a ready-to-use liquid medium (which is pre-filtered and in a presentation suitable for small volume fills). Where there are concerns about transmissible spongiform encephalopathies in relation to animal-derived products, alternative media like vegetable peptone broth (made from pea flour) can be substituted. Similarly, media fill runs with anaerobic media (alternate fluid thioglycollate medium without agar) can be required if anaerobes are isolated from the environments and product samples.
Not all culture media is of the same quality or suitable for use with a media fill. Important criteria for suitable media for an APS include [5]:
• Sterile
• Cold filterability
• Solubility
• Format (granulated media)
In considering these:
Sterility
Using a medium that has been pre-sterilized avoids the use of a medium that is contaminated and hence which could pose a challenge to the sterilizing filter. The sterilization process, typically gamma- or x-ray irradiation, should not affect the properties of the medium or affect microbial growth.
Cold filtration
Using a medium that requires heating to enable it to dissolve and for it then to cool to pass through a filter is not always practicable within manufacturing. In addition, the application of heat needs to be carefully applied otherwise the nutritive properties of the medium can be affected. Filterability is assessed in terms of the time taken for a given volume of media to pass through filters made of different materials (such as PES, nitrocellulose, PVDF etc.), without clogging or loss of flow characteristics.
Selecting a medium with cold filtration properties can help prolong filter life and reduce replacements, saving both time and expenditure.
Format
A base medium is typically available in powdered form. Whilst common, there are advantages to using media in a granulated form. These advantages result from the agglomerated larger particles and include reduced dust release and spread during preparation. This reduces health risks to operators and prevents environmental and cross-contamination. In addition, the lower generation of dust reduces deposits on equipment, and this also eases handling and cleaning.
Granulated forms also have longer shelf life and material stability compared with powders.
Solubility
In a busy manufacturing area, time spent waiting for media to dissolve is unproductive. Hence, using a medium with rapid and effective solubility reduces preparation time and effort. Granulation is a process that can improve solubility and accelerate the time taken to dissolve when compared to equivalent products in a power format. The filtered medium should be of a clear appearance.
In addition to the above, the method of transferring culture media into the facility must be conducive to good contamination control principles (such as with the medium being provided triple-wrapped by the supplier and with the user having a defined method for introducing the medium into the manufacturing area).
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Bacterial growth (designed by Tim Sandle)
Incubation and growth promotion
Media fill evaluation units should be incubated for not less than 14 days at temperatures between 20°C and 35°C. Many firms adopt a two-temperature incubation schedule to incubate at 20-25°C for a minimum of 7 days followed immediately by incubation at a higher temperature range of 30-35°C for a total minimum incubation time of 14 days. Other incubation schedules should be based on supporting data.
To be GMP compliant, the selected culture medium should be demonstrated for the growth promotion test. The microorganism panel is often the one used for the sterility test, as per pharmacopoeia guidelines. Some organizations include environmental isolates taken from the aseptic filling area by way of a more robust challenge. It is recommended that growth promotion is performed at the end of incubation in order to demonstrate that the incubation conditions were not detrimental to the growth of organisms. The selection of containers for testing should be performed in the manner of samples selected for sterility testing, that is randomly selected units equidistant through the batch. The method of performing growth promotion testing should be based on pharmacopeial guidance (such as using the organisms recommended for the sterility test qualification) and ISO 11133: 2014 [6] principles. It may be appropriate to increase the size of the test panel by including representative organisms based on a review of the manufacturing environment.
If growth promotion testing fails, the media fill test is invalid and an investigation must be performed. The media trial should be promptly repeated.
Summary
The APS remains a critical exercise to be conducted by the manufacturers of aseptically filled products. This article has discussed several considerations. Arguably, the two areas of greatest importance are with the design (especially with the capture of ‘worst-case’ factors when selecting the appropriate line/product combinations: longest fill duration; widest container opening and longest exposure time of open container) and with ensuring that media of appropriate quality is selected (including the considerations of sterility, and granulation, filterability).
References
1. Agalloco, J. and Akers. J. Risk Assessment and Mitigation in Aseptic Processing, 3rd edition, CRC Press, 2016.
2. Sandle, T. Designing Aseptic Process Simulations: The Time and Container Number Conundrum, Journal of GxP Compliance, 20. 1-12, 2016
3. Cundell, A. Microbial Testing in Support of Aseptic Processing, Pharm. Tech. 56-66 (June 2004).
4. Sandle, T., Leavy, C. and Needham, G. (2012). A Risk Matrix Approach for Media Simulation Trials, Journal of Validation Technology, 18 (4): 70-78
5. Sandle, T. (2018) Microbiological Culture Media: A Complete Guide for Pharmaceutical and Healthcare Manufacturers, DHI/PDA, Bethesda, MD, USA, ISBN Number: 9781942911159
6. ISO 11133:2014 Microbiology of food, animal feed and water — Preparation, production, storage and performance testing of culture media
Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)
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