Over the past decade, the emergence of advanced therapy medicinal products (ATMPs) has fundamentally changed the approach to developing, manufacturing, administering, and treating diseases. This paradigm shift is driven by allogenic and autologous cell or gene therapies (CGT). Although these advanced modalities show significant promise, they also introduce various challenges regarding scalable manufacturing, safety, and logistical considerations regarding patient administration. Regulatory agencies and industry experts have identified the unique challenges and requirements associated with ATMP production. In response, these groups have provided updated guidance regarding the safe manufacturing and testing of these products. 

In 2015, the European Pharmacopoeia (Ph. Eur.) published issue 27.3, which included Chapter 2.6.27, “Microbial Examination of Cell-based Preparations.” This chapter intended to address cell-based preparations or products where the examination for sterility cannot be applied.¹ The chapter addressed challenges associated with ATMPs and cell-based preparations, such as shelf-life limitations, sample size, and rationale for selecting an adequate test method using risk analysis. While some allogenic and autologous therapies are cryopreserved, many have a shelf-life of a few hours or a few days. The 14-day sterility test outlined in Ph. Eur. 2.6.1 is inadequate for these short-life products. Another challenge is the sample size and volume to be tested. Due to the manufacturing process and size of the production batch, the corresponding test sample is extremely limited. Ph. Eur. 2.6.27 addresses this challenge by providing specific guidance for cell-based preparations where the total inoculum volume is recommended based on the volume of the batch, whether it be <1 mL or up to 1L. The chapter also provides a rationale for selecting methods, including detection by alternative methods, such as Adenosine Triphosphate (ATP) Bioluminescence (Ph. Eur. 5.1.6), to provide test results.²

The United States Pharmacopeia (USP), which provides procedures and acceptance criteria for assessing medicinal quality, also addressed the complex ATMP challenges. In 2019, USP published <1071> “Rapid Microbial Tests for Release of Sterile Short-Life Products: A Risk-based Approach.” As an informational chapter, USP <1071> contains no mandatory tests, assays, or requirements. However, USP <1071> provides guidance on user requirements, risk-based monitoring, and release testing, and descriptions of candidate rapid microbiological methods (RMM) technologies. It includes references to testing requirements outlined in USP <71> “Sterility Tests.” The chapter gives examples where USP <71> is unsuitable for product release testing and provides guidance on the volume of samples tested while referencing Ph. Eur. 2.6.27 for batch sizes of less than 40 units.⁴ Following the user requirements specifications for RMMs, USP 〈1071〉 introduces technologies, such as ATP-bioluminescence, as a well-established, recommended, and suitable growth-based technology that can detect microbial contamination.⁴

Overcoming challenges of ATP-bioluminescence for microbial detection in cell-containing samples

ATP-bioluminescence is a technology that has been validated and included in multiple regulatory approvals for routine release of a range of drug substances and products, including small molecule, biologics, vaccines, and others. Expert discussion groups from the Parenteral Drug Association (PDA) published a technology application in 2020 regarding scientific rationale and guidance on the microbiological controls of cell therapies that include various candidate RMM technologies.⁵ This guidance includes ATP-bioluminescence as a technology that could be incorporated into a control strategy for in-process monitoring, timed pre-release testing, and final product testing of cell therapies.⁵

To fully utilize this RMM, a key limitation of ATP-bioluminescence had to be addressed. Some CGT therapies are comprised of human cells that produce their own ATP. Since these eukaryotic cells differ in composition from microbial cells, Charles River Laboratories developed a novel methodology for removing the product containing eukaryotic cells while keeping the microbial cells intact. This methodology allows for the successful detection of ATP from microbial contamination without the interference of product ATP.

Samples using this method are prepared in Fluid Thioglycollate Medium (FTM) and Tryptic Soy Broth (TSB) and incubated for a designated amount of time at specified temperatures. Post-incubation, a cell lysing, and concentration procedure is performed, followed by a reagent treatment to reduce background ATP. After processing the sample, an ATP-bioluminescence assay is run on a luminometer to detect microbial contamination in the cell-containing samples. 

Charles River Laboratories published a case study that describes this method in which Jurkat T-Cells were tested using ATP-bioluminescence.⁶ A Sample Effects, or Sample Interference study was run and the Relative Light Units (RLU) from the Product Sample was compared against a predefined positive cutoff threshold to determine if residual background ATP from the T-cells were successfully depleted. In addition, S. aureus and B. subtilis were inoculated into a Micro Control and Micro Sample to verify that the assay could discern microbial ATP from the T-cell ATP. The data in Figure 1 confirmed that the T-cells tested did not interfere with or enhance the assay, thus demonstrating the feasibility of the technology.⁹

Method validation for ATP-bioluminescence for testing cell-containing samples to satisfy current requirements 

Following the guidance of Ph. Eur. 2.6.27 and USP <1071>, users are expected to perform validation studies to implement alternative microbiological methods such as ATP-bioluminescence. To help reduce the validation burden, Charles River Laboratories commissioned studies in accordance with USP <1223> “Validation of Alternative Microbiological Methods”, Ph. Eur. 5.1.6 “Alternative Methods for Control of Microbiological Quality” and PDA Technical Report No. 33, Revised 2013 “Evaluation, Validation and Implementation of Alternative and Rapid Microbiological Methods” that satisfied criteria required for validating a qualitative ATP-bioluminescence method. 

Equivalency was a criterion tested by Charles River Laboratories using ATP-bioluminescence on leukapheresis samples (targeted cell density of 1.0 x 10⁷ cells / mL). Using a predefined positive cutoff threshold, the results of the 4-day ATP method were compared to a 14-day USP <71> sterility test run side-by-side, with samples tested in parallel. The study referenced USP <1223> and used “decision equivalence” by acknowledging the binary nature of the test results in the form of presence (positive) or absence (negative).⁸ A sample size of 120 samples per method, divided equally among the organisms at the 1 CFU inoculum level, was chosen to statistically calculate if the ATP test is non-inferior to the 14-day USP <71> Sterility test. A statistical evaluation was conducted by an independent expert.⁸ 

The results illustrated in Table 1 were used to calculate the lower one-sided confidence interval for the difference in detection probabilities. The acceptance criteria for a non-inferiority margin of Δ = 0.2 was recommended in USP <1223>. As the calculated difference in detection probabilities of -0.0553 is within the non-inferiority limit, the null hypothesis of inferiority can be rejected. Thus, the ATP method is statistically non-inferior to the USP <71> reference method. 

Charles River Laboratories is committed to working with sponsors regarding product compatibility and complete RMM validations. Product types in Figure 2 have been evaluated and were confirmed to be compatible with ATP-bioluminescence. 

The future of ATP-bioluminescence beyond August 2025

Recent updates to USP have been added to provide additional guidance for selecting and qualifying RMMs for short-life products. In February 2025, USP published an update to USP <1071> “Rapid Microbiological Methods for the Detection of Contamination in Short-life Products—A Risk-based Approach” and introduced General Chapter <73> “ATP Bioluminescence-Based Microbiological Methods for the Detection of Contamination in Short-Life Products”. 

According to USP <73>, the new general chapter intends to guide risk-based testing for detecting microbial contamination in short-life products and encompasses short shelf-life products and/or short manufacturing times where the product must be administered as soon as possible.⁹ The chapter outlines a variety of test instructions, such as: 

1. Culture Media and Incubation Temperatures 
2. Growth Promotion Test of Aerobes, Anaerobes, and Fungi 
3. Method Suitability Test 
4. Sample Interference Study 
5. Determination of the Incubation Time of the Product to be Examined
6. Test for Microbial Detection in the Product to be Examined 
7. Monitoring and Interpretation of Results 

A new addition to the final USP <73> guidance is the “Determination of the Incubation Time of the Product to be Examined” section. The instructions are intended to provide users with suitable incubation time based on data generated from method suitability with an added safety margin.⁹ Data is generated by using microorganisms inoculated at no more than 10 CFU into the culture media containing the product and set on a growth rate of relevant test strains.⁹ The method suitability section also outlines the importance of organism selection, and the microorganisms used should include compendial test strains and slow-growing and/or local isolates relevant to the product or manufacturing process.⁹ 

Another requirement stated in the updated publication is a prerequisite that the primary validation of the method as described in <1071> must be available.⁹ USP <1071> clearly states that if the RMM is not described in a USP general method chapter, then validation is required according to USP <1223>.⁴ If the RMM is described in a general method chapter, in addition to the primary validation, the respective USP chapter (e.g. USP <73>) would also apply.⁴ 

Charles River Laboratories has successfully satisfied all criteria as outlined in USP <1223>; therefore, users can leverage the pre-existing primary validation data and concentrate on satisfying the test requirements as outlined in USP <73>. The primary validation also includes using several slow-growing organisms and molds relevant to the CGT manufacturing environment. Although the primary validation can be utilized, users must refer to USP <73> and always include relevant environmental isolates in method suitability testing of any RMM system. 

The updated publication of USP <1071> and USP <73>, effective August 2025, has a significant impact on end users successfully implementing RMMs included in the general chapters. The validation burden has been significantly reduced and now allows manufacturers of compounded sterile preparations, nuclear medicines and ATMPs to have a simple and effective blueprint to include ATP-bioluminescence in their microbiological control strategies.⁴ The updated guidance also suggests a difference in approach between USP and the European Directorate for the Quality of Medicines & HealthCare (EDQM) regarding the use of RMM technologies. EDQM held a workshop in October 2024 to discuss a certification system for validating RMMs, with the goal of increasing the efficiency and effectiveness of the RMM validation process. While these approaches may slightly differ, the actions of these organizations demonstrate continued commitment to providing testing solutions to address the challenges encountered with emerging advanced therapeutics, thus enabling faster access to critical healthcare for patients.  

References

1. https://www.gmp-compliance.org/gmp-news/european-pharmacopoeia-chapter-2-6-27-microbiological-examination-of-cell-based-preparations-revised#:~:text=A%20whole%20series%20of%20drafts,2

2. 2.6.27 Microbiological Examination of cell-based Preparations 

3. https://www.usp.org/frequently-asked-questions/identifying-official-text#:~:text=From%20a%20compendial%20standpoint%2C%20General,it%20is%20numbered%20above%201000 

4. USP <1071>: Rapid Microbial Test for Release of Sterile Short-Life Products: A Risk-Based Approach 

5. Risk Assessment Approach to Microbiological Controls of Cell Therapies, PDA Journal

6. Demonstration of ATP-Bioluminescence for Detection of Microbial Contaminants in T-Cells Using Celsis Adapt™ – Charles River Laboratories

7. Protocol No. 21-074, Celsis Adapt™ Cell Equivalency Fact Sheet

8. USP <1223>: Validation of Alternative Microbiological Methods 

9. USP <73>: ATP Bioluminescence-Based Microbiological Methods for the Detection of Contamination in Short-Life Products 

Brice Chasey is an Associate Director, Product Management with over 15 years of industry QC microbiology experience including over 10 years supporting, validating, and developing alternative microbiological methods. Brice now manages the Celsis Rapid Microbial Detection product portfolio at Charles River Laboratories. He has a Bachelor of Science, Microbiology from Arizona State University and currently resides in Chicago, Illinois.