Evaluating, Validating, And Implementing NAT-Based Mycoplasma Detection Methods

By BioPhorum

Implementing alternative methods for adventitious agents can slash >90% of testing time and realize cost savings of up to $5 million per product. Alternative adventitious agent detection methods, or adventitious agent tests (AATs), have been available for many years, and their use has been advocated by global regulatory authorities — but their widespread adoption in the biotherapeutic industry has been slow. Many traditional compendial methods used for the detection of adventitious agents are laborious, difficult to automate to alleviate data integrity concerns, and can cause significant delays before results are obtained.

Previous work¹ has identified that reluctance to embrace these technologies could be attributed to breakdowns in:

• strategy and approach (e.g., unclear drivers),
• technical (e.g., regulatory acceptance),
• cultural (e.g., reservations around change technologies), and
• implementation and execution (e.g., inadequate validation strategy).

The Biomanufacturing Technology Roadmap² outlined the aspiration for a < two-day AAT for product release by 2026. However, as the biomanufacturing landscape has evolved to include advanced therapy medicinal products, many of which have a short shelf life, there is a clear need to embrace new technologies that provide an assurance of safety from adventitious agents in a much shorter timeframe.

To address some of the challenges identified as roadblocks to the implementation of alternative AATs, this article discusses best practice guidance for the validation of an alternative mycoplasma detection assay.

Approach To Adoption Of NAT-Based Mycoplasma Detection Methods: A 9-Step Framework

There are variations in the approaches taken by biopharmaceutical manufacturers to implement alternative, nucleic acid testing (NAT)-based mycoplasma detection methods. The presented framework breaks the process down into nine steps, from initial evaluation to final implementation of the method.

Step 1: Identify The Operational/Business Need

Due to the limitations of current culture and indicator cell culture-based methods, researchers are involved in the identification and development of more rapid and accurate mycoplasma tests, taking advantage of recent technological progress.

A mycoplasma contamination event in a biomanufacturing facility results in costly cleanups. Although there are differences between species, the small size, slow growth, and sometimes subtle effects of mycoplasma make these bacteria ideal for adopting early and fast detection strategies in biomanufacturing. The ability of mycoplasma to evade detection and grow in mammalian cell cultures creates risk, in some ways more like viral contamination than typical bacterial contamination.

Mycoplasma and viral contaminations in the biotechnology industry are rare, but when they do occur, they lead to costly investigation and decontamination measures, drug shortages, and damage to public confidence in the manufacturer. When biopharmaceutical manufacturers find mycoplasma in their cultures, they decontaminate after first taking a limited number of culture samples for specification and testing raw material samples to trace the source of the contamination. The most important advantages of NAT-based mycoplasma detection methods include high sensitivity and the capability to rapidly detect a wide range of mycoplasma species. These methods would also facilitate faster decision-making, minimizing the spread of contamination in facilities that often handle multiple products.

Step 2: Define The Application

Rapid-release testing for adventitious agents such as mycoplasma can be applied in place of current compendial test methods. There must be no evidence of mycoplasma contamination in test materials for release, so a qualitative assay that simply demonstrates the absence or presence of mycoplasma is sufficient. Minimally, the limit of detection for the rapid mycoplasma release test should be equivalent (not inferior) to the compendial method. Specificity is another important aspect of the method validation. The alternative mycoplasma detection method should have no gaps in specificity, or — if a gap exists — a rationale must be provided for why this gap will be accepted and a mitigation must be defined.

Step 3: Assess The Requirements

There are certain core requirements common to most applications. These requirements may be used to undertake an initial screening of the applications available and may be based on internal company expectations, regulatory requirements, industry best practices, market regulation, and other sources. Examples of core requirements for any application include:

• data integrity,
• laboratory information management system connectivity, and
• reduced complexity and analyst subjectivity.

Specific method application requirements provide a helpful tool for describing precise requirements and offer further benefits in later steps. Subsequent requirements regarding the throughput will be more specific to the exact need and application.

The intended use(s) of the test method should be considered early. Sample types tested will impact filing strategies and project complexities (e.g., release of drug substance or finished product release versus screening of raw materials). Additionally, separate sets of requirements may be created to capture long-term versus short-term needs.

Step 4: Compare Options And Technologies — Landscaping And Candidate(s) Selection

Once a user requirements specification has been defined, the focus shifts to finding the best technology solution that meets as many (ideally, all) of the requirements as possible. To do this, the user requirements specification is used as a filter to sort the possible solutions based on their ability to meet the requirements.

Defining the list of possible solutions is a critical step that must be conducted without preconceived notions or bias. The widest possible search should be undertaken to identify solutions that could meet the overall need. It is useful to include technologies that may not meet all requirements since shortcomings may be covered by justified risk mitigation or a second technology.

Step 5: Develop The Business Case — Technical, Quality, And Business Evaluation And Justification

The technical analysis conducted in the previous step enables the most suitable technology to be selected for implementation. While this analysis is essential, and a clear “best choice” may be obvious, it does not provide the necessary justification for expending time and resources on the implementation. The next critical step is to develop the business case for the implementation.

The broad impetus of step 1 through the current step 5 is the identification of a need, gap, or opportunity for which a technical solution could be the answer. This is a foundation for the business analysis but is insufficient on its own, for building a case for investment in the new technology. A case must be made that the investment has tangible benefits in the short, medium, and long term. Obvious costs include initial procurement, validation, labor, ongoing supply, regulatory filing (in the case of changes to existing licensed products), and ongoing maintenance. Less obvious but also relevant to financial decision makers is the risk of lost opportunity due to investment in one project at the expense of another. Costs and benefits should be accurately quantified to show stakeholders that a project is worthy of investment.

Step 6: Perform Proof-Of-Concept Studies/Feasibility Studies/Pre-Validation Studies

Proof-Of-Concept/Feasibility

Once the systems suited to the purpose(s) have been determined, it is recommended that proof-of-concept or feasibility studies are undertaken before proceeding with more comprehensive pre-validation and validation studies or even the purchase of the system. The main purpose of the feasibility study is to verify quickly that there are no technical incompatibilities between the system and the product samples to be tested.

Pre-Validation

Pre-validation studies should entail a more comprehensive assessment in response to the questions asked during the proof-of-concept/feasibility stage, address specific end user needs defined at this stage, as well as internal and regulatory requirements. The outcome of this stage should be to assess whether the technology is fit for purpose and validation can commence, whether any further development is required, or whether an alternative technology or method needs to be considered.

Step 7: Validate At Pilot Or Primary Site

This is a critical step. Below, we summarize two validation examples that represent potential study designs based on positive feedback from health authorities around the globe:

Case Study 1: Description Of The Real-Time PCR-Based Direct Detection Assay

The validation process for a real-time PCR-based direct detection mycoplasma assay by one company was separated into two main activities:

1. the primary method validation
2. the validation for the intended use (test for interfering substances/product-specific method suitability test).

Method validation demonstrates and documents that the alternative, real-time PCR-based mycoplasma detection assay is suitable for the application by showing equivalency (noninferiority) to the compendial methods. Method validation acceptance criteria were determined prior to initiation of testing and are based on the guidance about method validation of alternative, NAT-based mycoplasma detection methods in the Ph. Eur. Chapter 2.6.7 Mycoplasmas, 2.6.21 Nucleic Acid Amplification Techniques, and ICH Q2A(R1) Validation of Analytical Procedures: Text and Methodology.

The following parameters were examined in the validation:

• Specificity
• LOD (sensitivity)
• Robustness
• Precision

A comparability study and suitability test were also carried out. All had specific acceptance criteria determined.

Case Study 2: Mycoplasma Assay Validation Outline

The validation strategy and plan utilised by another company is aligned with Ph. Eur. 2.6.7, Mycoplasmas, for Nucleic Acid Testing (NAT) based methods, specifically on guidance for sensitivity specificity, (LOD), robustness and selection of species tested in validation (inclusion and exclusion panels) and with ICH, Q2 (R1) Validation of Analytical Procedures, according to the guidance on validation of limit tests for impurities. The testing protocol evaluated in the validation should include the entire testing workflow, from sample preparation through detection and results analysis. The validation approach is as follows:

Specificity

• Part 1: Assay is specific to target species and does not detect off-target species.
• Part 2: Detection of mycoplasma species at the defined LOD recovered from the test sample matrices included in  the scope of the validation.

LOD

For lot release testing applications, the LOD validated should be aligned with the Ph. Eur. 2.6.7 guidance of 10 GC or CFU/mL test sample. For in-process control testing applications, the LOD may be less stringent based on process-specific risk assessment. LOD testing can be executed with purified mycoplasma genomic DNA (per guidance in the BioPhorum URS), live mycoplasma cells or a combination of both.

Robustness

The assay supplier may have data that support robustness generated during development of the assay. Additionally, data generated by the user during testing protocol optimization and qualification may be applicable to robustness and may not require repeating during validation.

Experimental plan: The experimental design for robustness is more open-ended and should be aligned with site-specific requirements on robustness testing as part of analytical method development, qualification, and validation.

Additional case studies can be found in the full report on this topic.³

Step 8: Deploy Global/Company-Wide Qualification Of Additional Laboratories

For global companies, site-to-site harmonization is an important aspect of a method and/or technology implementation and helps ensure consistent, safe, and reliable release of product across the globe. Generally, it is advantageous to pilot at one facility when new systems and strategies for validation are used since:

• The regulatory perspective is evolving, so subsequent validation iterations can account for new expectations.
• Resource constraints may limit implementation at several sites at once.
• Subsequent sites can take advantage of strategy and document precedents.
• Lessons learned throughout the process can be applied after the initial implementation.

Step 9: Define Regulatory Filings And Implementation

International regulatory authorities have published guidelines to demonstrate that biological products intended for preventive or therapeutic clinical use and prepared in cell culture substrates must be free of mycoplasmas to ensure product safety, purity, and potency. Therefore, early detection of mycoplasmas is essential for robust processes in the manufacturing of biopharmaceutical and cell therapy products. Regulatory authorities have published legally binding documents with national pharmacopeias for mycoplasma testing. These documents define the methods and detailed test protocols that may differ among countries and products to be tested.

There is a specific difference between conventional culture-based and alternative NAT-based mycoplasma testing methods: the traditional compendial methods, such as the culture method and the indicator cell culture method, are considered the long-standing gold standard and, therefore, are widely requested in all pharmacopeias and are largely harmonized across countries.

The situation is very different for rapid mycoplasma testing methods like NAT. Even though many national pharmacopeias mention NAT as a valid mycoplasma testing method, there is little harmonization across countries regarding protocols or validation requirements. All countries require an appropriate validation and comparison with conventional mycoplasma testing methods. The European Pharmacopoeia provides the most detailed NAT validation guideline of all pharmacopeias in chapter 2.6.76. Four requirements must be met by NAT-based mycoplasma testing methods: limit of detection, specificity, robustness, and comparability. For full and generic validation of a NAT-based method, it is advisable to include additional parameters such as precision and cross-contamination.

Conclusion

The effects of mycoplasma contamination are devastating in the biopharmaceutical and cell therapy industry, as entire production batches must be discarded, and the manufacturing plant must stop production. The framework presented here enables faster adoption of rapid methods suitable for lot release testing. It has the potential to save >90% of virus testing time that, in turn, realizes significant cost savings per product.

This article summarizes a recent BioPhorum publication on the topic, which includes three case studies and a detailed user requirement specification for NAT-based mycoplasma detection methods. To read more, check out the full paper in A structured approach for the evaluation, validation and implementation of NAT-based mycoplasma detection methods.

References

1. Rapid detection of bacteria and viruses: justification, regulation, requirements and technologies — how can industry achieve broad adoption? BioPhorum, 2019. Available from www.biophorum.com/download/trm-rapid-detection-of-bacteria-and-viruses-how-can-industry-achieve-broad-adoption_-october-2019/
2. Biomanufacturing Technology Roadmap. BioPhorum, 2017. Available from www.biophorum.com/download/in-line-monitoring-and-real-time-release/
3. A structured approach for the evaluation, validation and implementation of NAT-based mycoplasma detection methods. BioPhorum, 2023. Available at www.biophorum.com/download/a-structured-approach-for-the-evaluation-validation-and-implementation-of-nat-based-mycoplasma-detection-methods/