Aseptic process simulation (APS) is a critical periodic test that manufacturers of aseptically filled products must conduct to verify that their process is capable of assuring product sterility. In this simulation, the actual product is typically replaced with a sterile nutrient medium. An APS should closely replicate the routine aseptic manufacturing process, including all the critical manufacturing steps. Given the significant variability in manufacturing processes, designing an effective APS is a complex task that requires careful consideration of the specific characteristics of the product and the methods employed in its production. As part of the contamination control strategy, the test design must be thoroughly documented. This ensures that the simulation can be executed and evaluated consistently, providing a reliable assessment of the aseptic manufacturing process’s integrity.
Regulations and guidance related to APS include the PDA Technical Report No. 22, the FDA’s ‘Sterile Drug Products Produced by Aseptic Processing - Current Good Manufacturing Practice’, and the EU GMP Annex 1 ‘Manufacture of Sterile Medicinal Products’, which is the most recently amended. According to Annex 1, an APS must be conducted under worst-case scenarios for various process variables such as line speed, container sizes, media volumes, and possible interruptions during manufacturing. Additionally, staff-related variables, including the number of operators, their working duration, and break times, must be considered.
EU GMP Annex 1 further specifies requirements regarding APS frequency and batch sizes. The culture medium should be clear and, whenever feasible, be filled into transparent containers that allow visual detection of turbidity, which would indicate microbial growth. Before incubation, the filled APS units should be agitated or inverted to ensure that the medium contacts all interior surfaces.
Growth promotion of culture media for APS
The medium must effectively support the growth of a scientifically justified group of reference microorganisms, including suitably representative local isolates. Samples of the filled units should undergo positive controls through inoculation with these strains. Due to the broad spectrum of organisms that may be present, a non-selective medium for mesophilic and aerobic growth is typically required; tryptic soy broth (TSB) is commonly used. The supplier should provide results of growth promotion tests with a panel of typical contaminant bacteria, yeasts, and molds. Some in-house strains may not be suitable for the APS panel, for example, isolates from water testing that require lower levels of nutrients to avoid nutrient shock in the complex medium. These strains should be tracked by other means, for example, the bioburden testing in combination with trending analyses. Anaerobic media are rarely used for APS except in specific cases, where the complete manufacturing process is anaerobic or when previous sterility tests have been positive for obligate anaerobes.
Many suitable media contain animal-derived materials, making it essential to ensure that they are free from bovine spongiform encephalopathy (BSE). We source the bovine ingredients of our TSB exclusively from countries classified by the World Organization for Animal Health as having a negligible BSE/TSE risk, and primarily from Category C tissues (no detectable infectivity). These media come with certificates that provide full traceability of all animal-derived material. As an animal-free alternative to TSB, we offer a vegetable peptone broth with identical specifications, including growth-promoting properties.
Culture media formats for APS
Most suppliers can offer their APS media in both a dehydrated and a ready-to-use format, typically triple-bagged in USP class IV material and, for example, gamma irradiated to make them suitable for cleanrooms. The choice between these formats is not always solely based on convenience but may also depend on which option better mimics the actual manufacturing process. Cold solubility is a particular advantage when using dehydrated media because sterile filtration avoids the need for autoclaving. Undissolved material can clog the filter, which should be avoided because changing it during an APS is time-consuming and increases the contamination risk. We have opted to offer our dehydrated APS media exclusively in a granulated format as this provides several benefits over powdered media, including better solubility and higher filtration speed, with no significant disadvantages. Tests are performed at various stages of the production process to verify these properties. Granulated media also generate considerably less dust, which can contaminate the lab or aseptic manufacturing area. Dust inhalation poses health risks, including toxicity and possible allergic reactions.
Ready-to-use, pre-sterilized culture media for APS typically come in 10-liter bags, equipped with a choice of connectors and a septum to inject additives. Our ready-to-use media are sterilized by autoclaving, followed by a two-step filtration down to a pore size of 0.2 µm. This also removes viable mycoplasmas, tiny bacteria that reach high titers without causing turbidity, which is why they are often difficult to detect after the typical 14 days of incubation for an APS. For our granulated APS media, we use gamma irradiation to ensure they are sterile and free from mycoplasmas, which we verify and state in the Certificate of Analysis.
Learn more about granulated and ready-to-use culture media for aseptic process simulations: SigmaAldrich.com/mediafills
Author Details
Dr. Anne-Grit Klees- Lead Expert, Product & Portfolio Manager, Environmental Monitoring, MilliporeSigma; Sabine Bessières Recasens- Regional Application & Commercial Tactics Manager, Industrial Microbiology, WEU, MilliporeSigma
Publication Details
This article appeared in American Pharmaceutical Review:Vol. 28, No. 3
April 2025Pages: 38-39
Subscribe to our e-Newsletters
Stay up to date with the latest news, articles, and events. Plus, get special
offers from American Pharmaceutical Review delivered to your inbox!
Sign up now!