How To Perform Stability Testing For Products With Short Shelf Life
What are stability, stability testing, and the importance of stability testing for medical products?
Stability is a suspended state where there is no change (or small changes that are happening internally at a consistent rate such that the net change moment to moment is zero). In terms of FDA regulations, stability testing covers five FDA categories: chemical stability, physical stability, therapeutic stability, toxicological stability, and microbiological stability of a medical device or product. Chemical stability refers primarily to drug formulations and ensures that all molecules in a formulation remain in their therapeutic state and do not undergo additional chemical reactions over time. However, medical devices must also be protected from oxidative chemical reactions or other chemical reactions that could cause the degradation of product materials. Physical stability refers to biological therapeutics (maintaining the physical integrity of protein structures over time) and the physical functional stability of medical devices or combination products. Therapeutic stability refers to the stability of a product or device’s therapeutic efficacy over time. Toxicological stability refers to the stability of the medical product, cosmetic, drug, or medical device’s toxicity levels over time. Finally, microbiological stability is the sterility of the product over time. Overall, stability testing ensures that medical devices and medical products are unchanging over time and keep patients safe. For medical and cosmetic stability testing, accelerated stability testing is used to simulate the long-term stability of a product in half the time. Accelerated stability testing is extremely advantageous for reducing timelines. However, accelerated stability testing does not prevent the need for real-time stability studies. Indeed, accelerated stability testing and real-time stability studies are often run in parallel so that the FDA receives stability testing data requirements as soon as possible. Products with short shelf life have additional challenges when it comes to stability testing for reasons detailed below. Thus, pharmaceutical and cosmetic stability testing may need to be modified for products containing biologics and have a short shelf life.
When is USP 71 unsuitable to use for stability testing, and which medical products qualify for rapid microbial testing?
The current growth-based sterility tests that follow USP 71 have an incubation period of at least 14 days. The test is not helpful for products with a shorter shelf-life or products prepared for immediate use that would be administered to patients before the traditional sterility test incubation time was complete. These short-life products include compounded sterile preparations (CSPs), positron emission tomographic (PET) products, and cell and gene therapies, which require rapid microbial tests, covered by USP 1071, instead of traditional stability tests from USP 71. Indeed, patient safety is best verified by a test that detects microbial contamination prior to product use. Various rapid microbial tests (RMTs) have different lengths of time to result, specificity, the limit of detection (LOD), and sample sizes. For example, due to the short half-life of radiotracers, most radiopharmaceuticals benefit from a real-time microbial test. On the other hand, CSPs and cell therapy products benefit from an overnight test or a microbial test completed within 48 hours. This article will cover USP 1071 guidelines on rapid microbial testing selection, test methods, and test requirements for short shelf-life sterile products. In most cases, cosmetic stability testing will be performed via accelerated stability testing and fall under traditional USP 71 testing methods.
Rapid Microbial Testing Requirements For Short Shelf-life, Sterile Products
The requirements for different rapid microbial testing technologies include:
- As short as possible time to result, ideally in real-time or less than 24 hours, preferably prior to the administration of the product
- Ability to detect, preferably less than 100 colony-forming units (cfu) in the test sample
- Ability to detect a wide range of viable microorganisms in a product
- Sample quantity, i.e., the minimum number of articles tested and quantity per container tested that does not consume a large proportion of the available product; whenever feasible, manufacturers should consider assay requirements during process design
- Aseptic test material handling, i.e., closed systems to reduce inadvertent contamination during testing
- Availability of instruments and reagents from multiple vendors
- Availability of reference standards, negative and positive controls, appropriate for technologies that use signals other than
- the colony-forming unit
- Ease of use and simplicity of test and data interpretation
- Low rates of false positive and false negative results
- A method suitability testing strategy for each specific product
- Improved patient safety arising from the completion of the test prior to administration and tests that provide progressive monitoring and reports of detection of sterility test failure
- Ability to identify the detected microorganisms, which may be helpful to the clinician administering the products and investigating to determine their source
- Robustness and reliability of equipment and reagents used in the testing
- Sample preparation suitable for both manual and automated methods
How are rapid microbial tests for products with short shelf-life performed?
Technologies recommended for rapid microbial testing are:
- Adenosine triphosphate (ATP) bioluminescence
- Flow cytometry
- Isothermal microcalorimetry
- Nucleic acid amplification
- Respiration
- Solid-phase cytometry
Adenosine Triphosphate (ATP) Bioluminescence
The energy from a living cell is stored as ATP. When exposed to luciferase, ATP energy can be measured as light (an enzyme that helps the American firefly glow). Each ATP molecule consumed by luciferase produces 1 photon of light. These photons of light are picked up by a luminometer and expressed in relative light units (RLU). For a rapid microbial test for sterile short-life products, an enrichment culture of the product for two to seven days is needed to read the microbial content. Otherwise, the microbial content may be too low for the threshold RLU, indicating a false negative result. Enrichment cultures can be performed in liquid media to reach a threshold ATP level or on a membrane filter on solid media to form microbial colonies. Cosmetic stability testing using ATP bioluminescence may be difficult to perform on lipid-based products such as ointments.
Flow Cytometry
Flow cytometry detects fluorescently labeled viable microbial cells after an enrichment culture step of one to two days. A fluorogenic substrate or a vitality stain (nucleic acid-specific stain) differentiates viable cells from dead cells and cellular debris. A laser illuminates each cell in the flow stream when performing the flow cytometry, and a dual photomultiplier array detects the emitted light. The light signal is digitized and interpreted by software. Cosmetic stability testing of lipid-based products may also be cumbersome using flow cytometry.
Isothermal Microcalorimetry
Isothermal microcalorimeters monitor heat changes in closed vials to determine microbial metabolic activity and growth. Two to seven days of microbial enrichment via culture is needed for active microbial cells to be detected.
Nucleic Acid Amplification
Real-time quantitative polymerase chain reaction (PCR) monitors the exponential phase of PCR through 36–48 cycles of amplification using universal primers and probes to estimate the initial quantity of microbial DNA. The microbial DNA quantity is proportional to the number of microbial cells in the test sample. For PCR, cellular RNA turns over rapidly and is a better indicator of viable microorganisms. RNA can be converted into a complimentary copy of DNA (cDNA) using the reverse enzyme transcriptase. Then the cDNA can be analyzed in real-time either with a quantitative (enumeration test) or qualitative assay (sterility test). The addition of a growth-based enrichment step for at least 24 to 48 hours and comparing the PCR results before and after cultural enrichment may provide additional accuracy for rapid microbial sterility testing. Direct comparison of growth-based and nucleic acid amplification-based assays is complicated because nucleic acid amplification-based assays detect both viable and non-viable organisms and are a measure of microbial genome copy number, not colony-forming units. Thus, a nucleic acid amplification-based assay should be defined in terms of genome equivalents per milliliter.
Additional advantages of non-growth based RMTs like nucleic acid amplification:
- With close to real-time testing, the test will be completed before the short-life products are infused into a patient
- Detection of culture-negative infectious agents
- The test is minimally affected by antibiotics in the test sample
The test is less sensitive to the background resulting from animal cell lysis (e.g., particles, ATP) than other technologies since specific microbial genes are targeted.
Respiration
Respiration technology ranges from respirometers to gaseous headspace analyzers to automated blood culture systems. Aerobic and anaerobic broth formulations allow for the recovery of most microorganisms responsible for bloodstream infections with a five-day incubation. Respiration has been successfully used for sterility test cell therapy products cells using a seven-day incubation. One respiration technology, the tunable diode laser absorption spectroscopy (TDLAS), can measure oxygen depletion or carbon dioxide increases in closed units containing growing microorganisms in a culture medium. The technology can be used for automatic media fill inspection. All the respiration systems require a certain level of microbial growth for metabolic activity to be detected. Thus, respiration RMTs take two to seven days to obtain results.
Solid Phase Cytometry
Instrumentation combining solid phase cytometry, fluorescent labeling, and solid-phase laser scanning allows for rapid counting of viable microorganisms in filterable liquid samples. For these solid-phase cytometry tests, microorganisms are collected by filtration on 0.45-micron polyester membranes and treated with background and viability stains. The stained filters are then scanned in a cytometer by a high-speed, 488-nanometer laser. The scan is displayed as a map and used to detect individual viable microorganisms in two to three hours. Most cell therapy products are non-filterable, so this technology may not be compatible with these types of products.
RMT Method Suitability
The suitability of the rapid microbial test must be demonstrated for each product tested. Each RMT technology has different signals for microbial growth ranging from colony-forming unit to fluorescent labeling to RNA/DNA quantities. Thus, the microbial results using different RMT technologies may not be equivalent. Method suitability testing will confirm that microbial results align with the microbial detection levels needed to verify products are safe for patient use.
Summary
Overall, short-life sterile products, such as compounded sterile preparations (CSPs), positron emission tomographic (PET) products, and cell and gene therapies, require rapid microbial tests covered by USP 1071 instead of traditional stability tests from USP 71. Rapid microbial tests are selected amongst five primary technologies: adenosine triphosphate bioluminescence, flow cytometry, isothermal microcalorimetry, nucleic acid amplification, respiration, and solid-phase cytometry. For cosmetic stability testing, lipid-based products (like ointments or lotions) present challenges for some rapid microbial tests. It is essential to consider the suitability of the rapid microbial test you perform for products with short shelf life. The microbial results can be obtained prior to patient administration. All in all, when selecting a contract testing organization, ensure you choose one that can support you with selecting appropriate stability testing for your unique medical device or product needs.
MycoScience is a contract manufacturing organization that specializes in filling sterile syringes and vials for parenteral products. MycoScience also offers Stability Testing, Bacterial Endotoxin Testing, Preservative Efficacy Testing, Sterilization Validations, Bioburden Testing, Cleaning Validations, Microbial Aerosol Challenge Testing, Accelerated Aging, Microbiology Testing, Cytotoxicity Testing, EO Residual Testing, Package Integrity Testing & Environmental Monitoring services for medical device companies, and allied industries. MycoScience is an ISO 13485 certified facility.
References
United States Pharmacopeial Convention. <1071> Rapid Microbial Tests For Release Of Sterile Short-Life Products: A Risk-Based Approach. Rockville, MD, USA. 2021. (USPC <1071>).
