Cell and gene therapies are transforming medicine and offering new hope for untreatable medical conditions. But keeping these therapies free from contamination can be challenging.
Unlike traditional drugs, they involve living cells that cannot be heat-sterilized or filtered without risking damage. This makes sterility testing critical and complex — even a minor contamination risk can jeopardize patient safety.
So, how do manufacturers strike the right balance between strict contamination control and preserving product viability?
In this blog, we’ll examine the unique sterility testing challenges in cell and gene therapy, explore strategies to improve accuracy, and explain how Biostate AI’s advanced RNA sequencing solutions support safeguarding these innovative treatments.
What is Sterility Testing?
Sterility testing determines whether a product, material, or biological sample is free from viable microorganisms such as bacteria, fungi, or other contaminants. It is a vital quality control step in pharmaceuticals, biotechnology, and medical devices to confirm product safety and efficacy.
Sterility Testing in Gene and Cell Therapies
Gene and cell therapies are advanced treatments designed to modify or replace genetic material or introduce new cells to help treat or prevent diseases. They hold great promise for conditions such as cancer, genetic disorders, and autoimmune diseases.
Since gene and cell therapies are made using living cells, it is crucial to ensure they are free from harmful microorganisms. Unlike traditional drugs, these therapies cannot undergo terminal sterilization.
| Terminal sterilization is a sterilization process applied to a product in its final sealed container to eliminate all viable microorganisms. This method is commonly used for pharmaceuticals, medical devices, and other sterile products.It typically involves methods such as:Heat (e.g., autoclaving)Irradiation (e.g., gamma rays)Chemical sterilants (e.g., ethylene oxide gas)Since terminal sterilization occurs after the product is sealed, it provides high sterility assurance. However, gene and cell therapies cannot undergo this process because the heat or chemicals would damage the living cells required for treatment. |
Therefore, they must be manufactured in strictly controlled environments using aseptic techniques (prevent contamination by maintaining sterility during product handling and manufacturing) to minimize contamination risks. Sterility testing plays a vital role in this process, confirming that the final product is safe for patient use.
When is Sterility Testing Performed?
Sterility testing is carried out at multiple stages to catch contamination risks early and confirm product safety before use. Timing is crucial, especially for therapies with short shelf lives. Testing too late may delay treatment, while testing too early could cause contamination to be introduced later in production. Key testing points to balance speed and accuracy are mentioned below.
- During Manufacturing: In-process sterility testing is often performed during production, particularly when open systems are involved, or prolonged processing increases contamination risk. This step helps identify contamination early, preventing issues from reaching later stages.
- Final Product Testing: A representative finished product sample must undergo sterility testing before release. Since cellular therapies are often administered quickly due to their short shelf life, sterility tests may run concurrently with product distribution, with results confirmed shortly after.
- Method Suitability Testing: Also known as qualification testing, it is performed before routine sterility testing begins. Method suitability testing is required to confirm that the cell or gene therapy product itself does not inhibit the detection of viable microorganisms. This step ensures accurate results, particularly when testing complex biological materials.
Effective sterility testing safeguards patient safety and prevents costly recalls and production delays. But what makes it so critical in the first place? Understanding its impact reveals why this step is essential for ensuring safe and reliable treatments.
Why is Sterility Testing Important?
The process of ensuring sterility in pharmaceutical products is crucial for patient safety. In 2022, sterility issues were among the top reasons for drug recalls, with 68 incidents leading to the removal of approximately 96.1 million units from the market.
Such recalls highlight the critical need for stringent sterility testing to prevent contamination and ensure the efficacy of treatments.
1. Safety Assurance
Cellular therapy products are designed to treat serious medical conditions, often in immunocompromised patients. Even minor microbial contamination can pose significant health risks. Sterility testing confirms the absence of harmful bacteria, fungi, and other microorganisms before these products reach patients.
2. Regulatory Compliance
Regulatory agencies like the FDA and EMA require sterility testing to ensure product safety. The United States governs this by the Code of Federal Regulations (Title 21, Parts 210, 211, and 600-680), with sterility testing standards specifically outlined in sections 610.12 and 610.30.
These regulations define testing requirements for both sterility and Mycoplasma contamination.
3. Risk Mitigation
Cellular therapies often involve complex manufacturing steps and open handling processes, increasing contamination risks. Sterility testing is a safeguard, helping manufacturers detect contamination early and preventing unsafe products from reaching patients.
4. Product Stability and Viability
Cellular therapies have a short shelf life because they are made from living cells that are highly sensitive to environmental conditions. Temperature, oxygen levels, and nutrient availability can impact cell survival and function.
Hence, rapid sterility testing methods help ensure the product remains safe without causing delays that could affect its effectiveness.
How is Sterility Testing Performed?
Sterility testing involves a combination of traditional and other alternative methods to ensure cellular therapy products are free from microbial contamination. Here’s an overview of the key techniques and important procedural steps:
Traditional Methods
Traditional sterility testing methods are widely used in pharmaceutical and biological product manufacturing to detect microbial contamination. The United States Pharmacopeia (USP) <71> outlines two key procedures— direct inoculation and membrane filtration.
Both methods are well-established, offering reliable results when performed correctly. The choice between direct inoculation and membrane filtration depends on factors such as product type, volume, and potential interference with microbial growth. Here’s how each method works:
- Direct Inoculation: Samples are introduced directly into growth media such as Tryptic Soy Broth (TSB) and Fluid Thioglycolate Medium (FTM). These media promote the growth of aerobic and anaerobic microorganisms, respectively. Incubation typically lasts 14 days, with visual inspections on days 3, 5, 7, and 14.
- Membrane Filtration: This method is ideal for larger volumes or products with antimicrobial properties. The sample is passed through a sterile membrane filter that traps microorganisms. The filter is then placed in TSB and FTM for incubation.
Alternative Test Methods
While traditional sterility tests are effective, they often fall short when applied to gene and cell therapies, where speed, sample volume, and complex product characteristics present unique challenges.
Alternative methods, such as Rapid Microbial Test Methods (RMMs), Blood Culture Systems, and ATP Bioluminescence, have emerged as reliable solutions that address these limitations.
- Rapid Microbial Test Methods (RMMs): These are advanced techniques that detect microbial contamination faster than traditional methods. Using technologies like nucleic acid amplification, flow cytometry, and automated growth detection, RMMs deliver results in hours or days instead of weeks.
| NoteNucleic Acid Amplification: Identifies microbial contamination by detecting and amplifying genetic material such as DNA or RNA sequences.Flow Cytometry: Uses laser-based technology to detect and count microorganisms based on their size, shape, and fluorescence markers.Automated Growth Detection: Monitors microbial growth through automated systems that track changes in factors like pH, CO2 production, or turbidity. |
They are ideal for time-sensitive therapies such as gene and cell treatments. When properly validated, RMMs offer reliable accuracy and are accepted by regulatory bodies like the FDA and EMA.
- Blood Culture Systems: Automated systems such as Bactec® and BacT/Alert® provide continuous monitoring and faster detection compared to traditional methods. These systems are widely accepted for sterility testing in cell and gene therapy manufacturing.
- ATP Bioluminescence: This method detects microbial activity by measuring ATP levels. While fast and highly sensitive, ATP bioluminescence is often combined with other techniques to ensure viable microorganisms are detected.
Key Procedural Steps
Sterility testing follows a structured process to minimize contamination risks and achieve reliable results. Key steps focus on proper sample handling, controlled incubation conditions, and verifying that the chosen method effectively detects microbial growth. Below are important procedural steps to consider:
Sterility testing requires careful sample preparation. Due to the risk of contamination, labs may preprocess samples before inoculation, particularly when membrane filtration is used.
- Sample Preparation and Method Selection
Sterility testing for gene and cell therapy products requires careful preparation to ensure accurate results. The chosen method depends on the product’s characteristics, such as volume, viscosity, and potential antimicrobial properties.
Direct inoculation is often suitable for small volumes or products that cannot be filtered. In this method, the sample is added directly to culture media. For larger volumes or products with antimicrobial agents, membrane filtration is preferred. This method allows rinsing steps to remove inhibitors and improve microbial recovery.
Proper sample handling and method selection are crucial for reliable sterility outcomes.
The direct inoculation method introduces the sample directly into two culture media– tryptic soy broth (TSB) and fluid thioglycolate medium (FTM).
TSB is incubated at 20-25°C, while FTM is incubated at 30-35°C to promote the growth of aerobic and anaerobic organisms, respectively. The incubation period typically lasts 14 days, providing sufficient time for slow-growing microorganisms to develop.
Throughout the incubation process, regular observations for turbidity are performed to detect potential contamination. This method is simple and effective, particularly for products that are difficult to filter.
Membrane filtration is a widely used technique for sterility testing, particularly for products with antimicrobial properties. The sample is passed through a sterile membrane filter with a pore size of ≤0.45 μm to trap microorganisms.
If the product has antimicrobial agents, the filter is rinsed with a sterile diluent three to five times to remove inhibitory residues. The membrane is then transferred into TSB and FTM and incubated under appropriate conditions for 14 days.
This method offers high sensitivity, especially for larger sample volumes, and is commonly used in cell and gene therapy product testing to ensure accurate sterility assessment.
Sterility testing in gene and cell therapies involves precise steps to ensure reliable results. However, achieving accuracy can be challenging due to unique product complexities and strict time constraints, requiring careful planning and method selection.
Challenges and Solutions in Sterility Testing for Gene and Cell Therapies
Sterility testing in gene and cell therapies presents unique roadblocks due to the nature of these products. Their short shelf life, limited sample volume, and complex manufacturing processes make traditional sterility tests less practical.
Contamination risks can arise at multiple stages, yet delays in testing may impact treatment timelines. Addressing these challenges requires careful planning and alternative methods that deliver faster, reliable results without compromising safety.
- Time Sensitivity
Gene and cell therapies often have short shelf lives, making it challenging to wait for traditional sterility test results, which can take up to 14 days. This delay risks product expiration before confirming its safety.
Solution: Rapid detection methods like RMM and automated blood culture systems can provide faster results without compromising accuracy.
- Complex Product Matrices
Cell therapies often contain concentrated cell mixtures or turbid solutions that can mask microbial contamination. This complexity increases the risk of false negatives, making standard methods less effective.
Solution: Techniques such as sample dilution, filtration, or combining multiple detection methods can improve test accuracy.
- Regulatory Acceptance
New sterility testing methods must meet strict validation standards to ensure they match the accuracy of traditional compendial tests. Without proper validation, regulatory approval may be delayed.
Solution: Conducting robust validation studies and engaging with regulatory agencies early can help streamline approvals.
Sterility testing is essential for ensuring the safety and effectiveness of gene and cell therapies. By combining traditional and rapid testing methods, manufacturers can maintain product quality, reduce contamination risks, and comply with regulatory requirements. Investing in validated rapid testing systems enables timely product release without compromising patient safety.
To Summarize
Understanding the complexities of sterility testing is crucial for maintaining the safety and success of gene and cell therapies. Reliable detection methods not only protect patients but also provide researchers with the confidence to move forward with their studies.
RNA sequencing plays a valuable role in this process. By analyzing gene expression patterns, researchers can identify unexpected microbial activity, contamination risks, or cellular stress, which impact sterility and treatment outcomes. This deeper molecular insight supports more accurate decision-making, particularly in therapies where cell viability and integrity are critical.
Biostate AI helps researchers achieve these insights with its comprehensive RNA sequencing services. Offering solutions for mRNA, lncRNA, miRNA, and piRNA analysis, Biostate AI enables data collection from a wide range of sample types, including FFPE tissue and small blood volumes.
Our cost-effective pricing — starting at $80 per sample for total RNAseq — makes advanced insights more accessible. For those seeking enhanced data analysis, our $250 per sample service simplifies the path from sample to publication, giving researchers the tools to uncover meaningful findings while saving time and resources.
Reach out to Biostate AI now to get your quote and take the next step toward safer, more effective research.
FAQs
- How does sterility impact the success of gene and cell therapies?
A: Sterility is crucial for the success of gene and cell therapies, as contamination can severely affect the safety and efficacy of treatments. Infected products can cause severe immune reactions or introduce pathogens into patients, compromising therapeutic outcomes. Ensuring sterility throughout production is essential for preventing such risks and meeting regulatory standards.
- Can sterility testing with RNA sequencing be performed on small samples?
A: Yes, sterility testing with RNA sequencing can be performed on small or limited samples, such as FFPE tissue or just 10µL of blood. Biostate AI’s RNA sequencing technology is optimized for low RNA yields, ensuring high-quality results even from small or difficult-to-process samples. It is ideal for testing gene therapies and cell-based products.
- How does RNA sequencing help detect contamination in cell therapies or gene therapies?
A: RNA sequencing helps identify contamination by detecting microbial RNA in a sample. If contaminants such as bacteria, fungi, or viruses are present, the sequencing process will pick up their RNA signatures. This allows for precisely identifying potential contamination without relying on traditional culturing methods, which may miss slower-growing or non-culturable organisms.
Sources
PubMed Central: J Clin Microbiol.: 6.1