Sterility testing represents a critical step within the overall contamination control strategy (CCS) for sterile products. Unlike environmental monitoring, in-process controls, or other types of testing, sterility testing is performed at the end of the manufacturing process and relies on a limited sample size. As a result, the conditions under which sterility testing is performed carry a heavy weight during audits and investigations. In this Pharma Matters Q&A, Amina Rahmoune, Laboratory Operations Manager, Sterility Assurance, Nelson Labs, explains how sterility testing in cleanrooms and isolators must be evaluated in the context of how effectively these environments support contamination control, not simply whether they meet minimum environmental classifications.

To support a comprehensive CCS, Rahmoune tells Contract Pharma, sterility testing environments are expected to align with manufacturing controls, personnel practices, and monitoring programs. European Union (EU) Good Manufacturing Practice (GMP) Annex 1 (2022) states that contamination control should be holistic, lifecycle-based, and documented, and should extend beyond manufacturing and take into consideration quality control activities, such as sterility testing. Sterility testing in traditional cleanrooms relies on aseptic techniques by operators, gowning practices, cleaning and disinfection, and environmental monitoring to maintain control. While this approach remains highly suitable when properly designed and managed, it introduces variability that must be actively mitigated through training, trending, and proceduralized practices (Parenteral Drug Association Technical Report No. 1). In contrast, sterility testing in an isolator reduces reliance on human factors by creating a physical separation between personnel and the test environment, which aligns closely with EU GMP Annex 1’s emphasis on eliminating contamination risks at the source rather than managing them procedurally.

According to Rahmoune, sterility testing environments must ensure aseptic handling in accordance with United States Pharmacopeia (USP) <71>, “Sterility Tests.” Multiple CCSs can be implemented when it comes to providing an aseptic environment for sterility testing. While both isolators and cleanrooms are widely used, regulatory expectations and operational priorities continue to evolve for each. To properly evaluate both approaches, manufacturers need to determine the most appropriate option based on the product, regulatory guidance, CCS considerations, risk, scalability, and automation trends.

As regulatory scrutiny continues to increase, sponsors are expected to demonstrate not only compliance, but also intentional choices in their sterility testing strategy. Auditors can ask for rationale as to why a particular approach was selected, how it fits within the overall CCS, and whether it would remain appropriate as products scale and move toward commercialization. This shift has moved decision-making away from historical practices or available infrastructure and toward approaches based on risk assessment. Sterility testing in cleanrooms versus isolators can often be viewed as interchangeable options; however, each represents a different risk within a CCS. Understanding those differences and how they affect sterility assurance, deviation rates, scalability, and audit readiness is essential for sponsors navigating the current regulatory landscape.

Contract Pharma: When weighing the merits of isolators versus cleanrooms for sterility testing, how are the expectations evolving for each approach?

Amina Rahmoune: As far as regulatory expectations are concerned, USP <71> requires sterility testing to be performed under aseptic conditions to prevent contamination. Chapter 71 does not prescribe a specific facility type, but rather emphasizes environmental control and procedural rigor. Industry guidance and EU GMP Annex 1 increasingly recognize isolators as a means of enhancing contamination control through the physical separation between personnel and the test environment and through validated decontamination cycles. However, both isolators and cleanroom environments remain acceptable when appropriately validated and monitored.

CP: When do isolators make more sense versus cleanrooms?

Rahmoune: Isolators are advantageous for minimizing false positives and preventing operator-related contamination. The enclosed design and validated decontamination cycles of isolators, which commonly use vaporized hydrogen peroxide (VHP), reduce environmental variability and contamination risk. Cleanrooms remain suitable when their associated infrastructure supports International Organization for Standardization (ISO) Class 5 conditions and when procedural controls effectively mitigate potential contamination risks.

CP: What product types, batch sizes, and lifecycle stages are more suitable for isolators or for cleanrooms?

Rahmoune: Pharmaceutical and aseptically processed products, such as injectables, biologics, and advanced therapies, where sterility failures have significant financial and patient impact, often benefit from isolator testing. Smaller batch sizes or high-potency products may also favor isolators due to containment advantages. Regarding product size, cleanroom testing is suitable for a variety of sizes. Cleanrooms may also be more suitable to support sterility testing for larger batch operations. Isolator testing, on the other hand, offers limited space and, therefore, may not accommodate larger product configurations. Additionally, because isolators rely on VHP decontamination, sample packaging must be compatible with the process. Specifically, packaging must demonstrate resistance to hydrogen peroxide ingress to ensure that residual sterilant does not compromise product integrity or interfere with sterility test outcomes. Furthermore, isolator testing is not appropriate for products tested using open funnel filtration methods, as it requires a fully closed system for filtration test methods.

CP: What should be considered for commercialization and scalability?

Rahmoune: As products advance toward commercialization, scalability and reproducibility become increasingly important. Isolators offer modular expansion and consistent environmental control, which may help reduce sterility-test failures and deviations. If testing is performed in-house, cleanroom expansion can be limited by the existing build and infrastructure, as cleanroom expansion would typically require HVAC modifications and broader facility upgrades. Some modular cleanroom designs can offer flexibility in expansion, although infrastructure remains a consideration when assessing scalability. Manufacturers and testing facilities should evaluate long-term throughput needs and risk-mitigation strategies.

CP: Can you compare both methods with respect to speed, risk tolerance, and regulatory expectations?

Rahmoune: Isolators may offer faster setup and reduced turnaround time due to controlled internal environments. They typically present lower contamination risk because they minimize human intervention. Cleanrooms depend heavily on operator technique, gowning discipline, and environmental monitoring. Regulatory expectations focus on demonstrated aseptic control rather than mandating one approach over another.

CP: How does the recent focus on automation and digital tools impact the decision-making process both short-term and long-term?

Rahmoune: Automation and digital environmental-monitoring systems enhance data integrity and traceability. Isolators often integrate automated decontamination cycles and continuous monitoring systems, supporting compliance with data integrity and good documentation practices (GDPs). Annex 1-compliant cleanrooms also benefit from continuous particle monitoring and electronic documentation systems conforming with 21 CFR (Code of Federal Regulations) Part 11.

CP: What are the most crucial factors to consider for sponsors faced with this decision?

Rahmoune: Sponsors should consider, along with packaging compatibility, their product type and how the approach to sterility testing fits within their CCS. Additionally, sponsors should prioritize regulatory compliance with USP <71> and Annex 1 requirements when selecting a sterility testing partner. A documented risk-based assessment should justify the selected approach, demonstrating consistent control of potential extrinsic contamination and alignment with current regulatory expectations for CCS.

For Further Reading

United States Pharmacopeia. (2024). USP <71> Sterility Tests.

United States Pharmacopeia. (2023). USP <797> Pharmaceutical Compounding—Sterile Preparations.

United States Pharmacopeia. (2024). USP <1116> Microbiological Control and Monitoring of Aseptic Processing Environments.

European Commission. (2022). EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use, Annex 1: Manufacture of Sterile Medicinal Products.

International Organization for Standardization. (2015). ISO 14644-1: Cleanrooms and Associated Controlled Environments — Classification of Air Cleanliness by Particle Concentration.

International Organization for Standardization. (2015). ISO 14644-2: Cleanrooms and Associated Controlled Environments — Monitoring to Provide Evidence of Cleanroom Performance Related to Air Cleanliness by Particle Concentration.

Parenteral Drug Association. (2011). Technical Report No. 34: Design and Validation of Isolator Systems for Aseptic Processing.

Parenteral Drug Association. (2022). Technical Report No. 90: Strategy for the Implementation of Contamination Control.