Setting the Standard Cleanliness Grades Behind Every Sterility Test Isolator

In contrast, manufacturing-focused documents, such as the recently revised EU GMP Annex 1: Manufacture of Sterile Medicinal Products provide detailed and stringent requirements for cleanroom classification and operations, including the location of closed isolators in Grade D and open isolators in Grade C environments, and understandably so, as any lapse in manufacturing control could directly compromise patient safety and product quality.

Although sterility testing and aseptic manufacturing serve different purposes, the lines between their requirements have become increasingly blurred. Sterility testing serves as a quality control measure to support product release decisions. A positive result (sterility test failure) may delay or prevent product release, but does not directly impact patient safety. In contrast, a false negative could have direct consequences for patients, although the controlled test environment has limited influence on this risk. Also, failures in aseptic manufacturing can immediately jeopardize patient health, underscoring the need for more stringent environmental and procedural controls.

The debate centers on the interpretation and perhaps excessive application of manufacturing principles to sterility testing environments.

While there is growing consensus that sterility testing should align closely with aseptic manufacturing practices, it is also important to distinguish between best practices and regulatory or mandatory requirements.

Regulatory Requirements

Drawing on input from multiple stakeholders from a range of regulatory perspectives, we conclude that there is currently no universal, mandatory requirement to locate a sterility testing isolator in a formally qualified cleanroom (i.e., Grade D). Instead, this remains a matter of rationale/justifiable risk assessment, subject to assessment and acceptance of the relevant competent authority.

That said, aligning sterility testing environments with those used in aseptic manufacturing, especially regarding isolator background classification, can simplify root cause investigations during a sterility test failure and streamline regulatory discussions with authorities (see Table 1).

We recognize, however, that this position on environmental classification for the background in which a sterility testing isolator is located may evolve. Regulatory guidelines are frequently revised, and with the increasing adoption of isolator technology in sterility testing, it is likely that more specific, harmonized guidance on this topic will emerge.

Comparison Between Isolators Used for Sterility Testing vs. Manufacturing Isolators

Isolators for sterility testing and isolators for aseptic manufacturing are both designed to maintain aseptic conditions but differ significantly in their applications and regulatory requirements. Understanding these differences is critical for regulatory compliance and optimizing facility design. The most important distinctions are listed in Table 2.

Sterility Test Result Errors

Examining the sterility test itself, which has a binary outcome (microorganisms present or absent), there are two basic types of errors that can occur: false positives and false negatives.

False positive result: A false positive result means that the test detected microorganisms in the units, even though the units were actually sterile.

False negative result: A false negative result means the test detected no microorganisms in the units, even though the units were actually non-sterile.

Examination of the False Positive Result

A false-positive result can be caused by (secondary) contamination of the units prior to or during testing, or by an incorrect readout (e.g., non-microbiological turbidity).

The false-positive result caused by incorrect readout can be readily revealed during investigation, when a subculture of the presumed microbiological contamination is not possible. The cause for the non-microbiological turbidity must then be identified (e.g., protein precipitates).

A secondary (microbiological) contamination of the units caused by improper handling of the testing units prior to or during testing cannot be distinguished from an actual unsterile testing unit. The type of microorganism identified in both cases will most likely be derived from the environment surrounding the isolator or human skin. If no obvious error occurred during handling or testing, the result must be counted as valid, and actual contamination of the units during manufacturing must be addressed, along with actions regarding batch release.

Therefore, potential sources of secondary contamination must be assessed, identified, and reduced to a minimum.

Sources of secondary contamination prior to testing could include improper cleaning or disinfection of the isolator or the samples to be tested, or an improper loading procedure. Potential errors could prevent H₂O₂ from reaching the surfaces and, consequently, prevent proper inactivation of microorganisms on them.

To contain these errors, various parameters should be validated and ensured. Points to be considered should include:

• Qualification of the loading pattern
• Qualification of the decontamination cycle
• Analyst qualification concerning cleaning and loading of isolators
• Adequate gowning of analysts during cleaning and loading process
• Detailed description of the cleaning and loading process (SOP)

Sources for secondary contamination during testing in a closed isolator could be failures of the sterility barrier itself. These could be due to an HVAC system failure, incorrect overpressure, or holes in isolator gloves.

In order to mitigate these errors, the following points should be ensured:

• Periodic maintenance and requalification of the HVAC system and the isolator itself
• Regularly integrity tests of the gloves and determination of intervals for replacements
• Operating the isolator in an overpressure mode if possible

To ensure an aseptic testing environment, an adequate environmental monitoring program should be occurred.

Investigation in case of a positive Sterility test result

The USP (chapter <71>) and the European Pharmacopoeia (chapter 2.6.1) clearly define only four points that could lead to test invalidation in the event of turbidity in the test vessels.

Therefore, only in very specific cases is it possible to identify a false positive sterility test result, which would allow a repetition of the test. In all other cases, the manufactured batch would have to be rejected.

In summary, the errors for a false positive sterility test result can be seen as a secondary contamination due to improper preparation of the isolator or the test sample. A higher level of cleanliness within the laboratory helps to lower the microbiological burden in the room. In order to reliably avoid secondary contamination, another focus must be placed on cleaning and disinfecting the isolator, the consumables and the samples.

A false-positive result does not imply a quality risk or even a risk to patient safety, as the batch would be rejected. It can therefore be solely seen as a business risk.

Examination of the False Negative Result

A false negative result can be caused by an inhibition of the growth-promoting properties of the media (e.g., due to residues of H2O2 or the product), accidental inactivation of the contamination (e.g., by H₂O₂ entering the product), or due to incorrect read-out (e.g., insufficient microbiological turbidity).

• The false negative result caused by incorrect read-out can be identified during method suitability testing, where the turbidity of the inoculated samples and, therefore, the method itself must be sufficient for read-out.
• A false negative result due to the product's antimicrobial properties can also be identified during method suitability testing, and the method must then be adapted accordingly to obtain valid results.
• A false negative result due to H₂O₂ residues is mitigated by integrity testing of the primary packaging materials for the test samples (vial/syringe and stopper) and the consumables. A H₂O₂ permeability study using spiked testing units also should be performed.

In summary, the errors associated with a false negative test result can only be mitigated by proper method development during method suitability testing and integrity testing of the primary packaging.

A general increase in cleanliness within the laboratory does not prevent false-negative results.

Assessment

The requirement for a Grade D background derived from Annex I can be seen as not mandatory to sterility test isolators placed in a quality control laboratory. This is also supported by several other guidelines, e.g. USP <1208> and WHO TR No. 961.

In general, Grade D environment as well as a CNC environment allows microorganisms, and it only shows the microbiological quality of the room itself. At least as important as the room is the cleanliness of the test samples, the consumables and the isolator chamber. Therefore, establishing this cleanliness area as a background for sterility test isolator and implementing environmental monitoring does not per se prevent false positive results in a sterility test.

One of the main driver for a valid result remains the preparation of the isolator including a correct disinfection of equipment and material as well as following the validated loading pattern.

Sampling, sample transportation to the QC lab, sample handling in the lab, preparation of the isolator and gowning in the QC lab must also be taken into account. To minimize all potential contamination risks safely, a CCS is recommended.

While there is no explicit requirement for sterility test isolators to be located within a classified cleanroom, the fundamental principles of cleanroom design should be considered and adapted to ensure a cleanliness level suitable for sterility testing. The following aspects should be determined based on a risk assessment taking into considerations the sterility test process and the design of the isolator:

• Access control
• Pressure cascade
• Airlock for personnel and material
• HVAC, temperature, humidity
• Non-viable particles
• Gowning procedure
• Environmental monitoring
• Cleaning/Disinfection

Based on consultations with several sterility testing experts, the consensus is that some pharmaceutical microbiology testing laboratories locate their sterility testing isolators in a classified environment to minimize business risks and preempt potential scrutiny, given varying interpretations of the requirement. However, others have adopted an alternative approach, supported by robust risk assessments, to justify the background classification of their sterility test isolators. Both practices reflect differing but valid interpretations within the regulatory framework and should be considered in shaping future guidance.

References

1. European Commission, The Rules Governing Medicinal Products in the European Union Volume 4. EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use, Annex 1 Manufacture of Sterile Medicinal Products (2022)
2. United States Pharmacopoeia, USP-NF 2025, <1028> Sterility Testing - Validation of Isolator Systems. The United States Pharmacopeial Convention, Rockville, MD
3. United States Pharmacopoeia, USP-NF 2025, <71> Sterility Tests. The United States Pharmacopeial Convention, Rockville, MD
4. European Pharmacopoeia (Ph. Eur.) 11th edition, 2.6.1. Testing for sterility.
5. European Pharmacopoeia (Ph. Eur.) 11th edition, 5.1.9. Guidelines for using the test for sterility.
6. US. Food and Drug Administration. Guidance for Industry. Sterile Drug Products Produced by Aseptic Processing - Current Good Manufacturing Practice (2004)
7. PIC/S PI 014-3. Isolators used for Aseptic Processing and Sterility Testing (2007)
8. Australian Government, Department of Health and Ageing, TGA guidelines for sterility testing of therapeutic goods (2006)
9. PDA Journal of Pharmaceutical Science and Technology, Technical Report No. 34. Design and Validation of Isolator Systems for the Manufacturing and Testing of Health Care Products (2001)
10. DIN EN ISO 14644-01:2015. Cleanrooms and associated controlled environments – Part 1: Classification of air cleanliness by particle concentration
11. World Health Organization, WHO Technical Report Series, No. 961, 2011