The European Pharmacopoeia 12.2 has updated the general chapter 2.6.7 Mycoplasmas, which will take effect on April 1, 2026. Compared with the previous version (released in January 2008 and revised in June of the same year), the new edition has undergone comprehensive modernization, structuring and content expansion, making it more in line with current biopharmaceutical industry practices and regulatory expectations, especially for Advanced Therapy Medicinal Products (ATMPs). The core revised or highlighted contents in the new edition are as follows:

01 Expansion of Structure and Scope

• Clear definition of scope 

The new edition clearly defines the test targets as Mollicutes (commonly known as mycoplasmas) at the beginning, and lists genera including Mycoplasma, Mycoplasmoides, Mesomycoplasma, Metamycoplasma, Mycoplasmosis, Ureaplasma, Acholeplasma and Spiroplasma. The previous version only generally referred to them as "mycoplasmas".

• Introduction of risk assessment principle 

The new edition emphasizes that method selection shall be based on risk assessment, taking into account factors such as culture medium, manufacturing process, product type and potential contaminant species. This is a fundamental conceptual shift from fixed procedures to science-based risk management.

• Clear sampling requirements 

The new edition adds and emphasizes that since mycoplasmas may adhere to or reside inside cells, both cells and supernatant shall be sampled for testing whenever possible.

• Addition of inhibitor testing 

In addition to retaining inhibitor testing in the culture method, inhibitor testing has been newly added to both the indicator cell method and the nucleic acid method, with specific protocols and test contents specified.

02 Nucleic Acid Amplification Technology (NAT)

• Clear status 

The new edition states at the beginning that "This chapter describes three methods for the detection of mycoplasmas: the culture method, the indicator cell culture method and nucleic acid amplification technology (NAT)", and further clarifies that "After appropriate validation, NAT may be used as an alternative to one or both of the above methods".

• Detailed classification 

The new edition subdivides NAT into three application modes:

• Direct NAT

• Cell-based enrichment followed by NAT

• Media-based enrichment followed by NAT

• Specific validation requirements 

The new edition provides extremely detailed validation guidelines for NAT, including:

• Clear comparability criteria: sensitivity of ≤10 CFU/mL as an alternative to the culture method; sensitivity of ≤100 CFU/mL as an alternative to the indicator cell method.

• Reference standards: introduction of the WHO International Standard (IS) for mycoplasma DNA as a calibration tool.

• Strain characterization: clear requirements for the preparation and characterization of strains used for validation. For viable reference materials, both Colony-Forming Unit (CFU) and Genome Copy (GC) are determined, and the acceptance criterion for GC/CFU ratio (<10) is defined, making NAT results more comparable and reliable.

• Limit of detection: a series of dilutions shall be performed using a panel of characterized and calibrated strains. Justification shall be provided if only DNA standards are used.

• Inhibitory substance testing: specific requirements for inhibitory substance testing are proposed for NAT, with test protocols exemplified in the validation guidelines.

03 Optimization of Culture Method and Indicator Cell Culture Method

• Applicability of culture conditions clarified 

The culture method and indicator cell culture method are only applicable to strains with an optimal growth temperature of 35–38°C; separate conditions may be required for unmentioned product-related strains or other cell production systems such as insect, fish and plant cell lines.

• Inhibitory substance testing 

The new edition retains the inhibitory substance testing requirements of the previous version in the culture method, and newly adds inhibitory substance testing in the indicator cell method, emphasizing that inhibitory substance testing is product-specific and shall be repeated after process changes. The new EP 2.6.7 has different focuses on treatment strategies for inhibitory substances in the culture method and indicator cell method:

① Treatment of inhibitory substances in the culture method

Main strategy: Dilution Principle: Reduce or eliminate the inhibitory effect by lowering the final concentration of the test product in the culture medium. Validation: The adopted dilution protocol must be proven to effectively neutralize inhibition by repeating inhibitory substance testing. Other possible measures: The new EP also mentions "or other measures (such as passage in a medium free of inhibitors)", i.e., subculture in an inhibitor-free matrix. This is rarely used in routine testing and is more of a theoretical alternative. Summary: Dilution is the preferred, most direct and commonly used treatment for the culture method.

② Treatment of inhibitory substances in the indicator cell culture method

Main strategy: More diverse, core to protect cells and/or mycoplasma-cell interaction

• Dilution:

Also applicable! The new EP clearly states that "If dilution of the sample is carried out, a larger volume of medium may be used or the inoculum volume distributed over several cell culture vessels." Consistent with the culture method, it reduces product concentration to mitigate cytotoxicity or interference with mycoplasma adhesion.

• Neutralization:

Specifically for viral product scenarios. Principle: If the test product is a viral suspension with cytopathic effect (CPE), the virus itself will kill indicator cells and make observation impossible. In this case, antiserum specific to the virus but without inhibitory effect on mycoplasmas can be used to neutralize viral infectivity, thus protecting indicator cells. Key point: "Neutralization" here is a very specific operation, referring exclusively to counteracting viruses with antiserum, not a general treatment for all inhibitory substances.

• Other alternative methods:

The new EP mentions that alternative methods such as "testing control cells" can be adopted in certain complex cases, but these methods must be fully justified and approved by the regulatory authority.

04 Updated Concepts and Terminology

• Flexibility of the combined use principle 

The previous version stipulated that two methods "must be used conjointly". The new edition changes it to "should be used conjointly", but adds an important exception clause: "unless otherwise prescribed in the monograph, or justified by risk assessment and authorized by the competent authority", ensuring the detection of both "cultivable" and "non-cultivable" mycoplasmas. This reflects regulatory flexibility and science-based decision-making principles.

• Emphasis on "validity" 

The new edition more clearly defines various conditions for invalid test results in the result interpretation section, strengthening the concept of quality control.

Compared with the previous version, the core changes of the new EP 2.6.7 are:

① Shift from fixed procedures to risk-based approach: introduction of risk assessment as the basis for method selection.

② Full embrace of NAT technology: upgrading NAT to a core method and establishing a comprehensive, rigorous and quantifiable validation and application framework for it.

③ More comprehensive and precise content: more accurately defining the scope of mycoplasma detection (e.g., Spiroplasma), refining operational requirements and clarifying sampling strategies.

④ Modernized language and structure: clear text structure, rigorous logic, accurate terminology, and better compliance with current GMP and regulatory science requirements.

These revisions reflect the development of the biopharmaceutical industry in recent years, especially in the fields of cell banks, viral vectors and Advanced Therapy Medicinal Products (ATMPs), as well as the urgent demand for rapid, sensitive and reliable mycoplasma detection methods.

Compared with pharmacopoeias of other countries, EP 2.6.7 included the NAT method at an earlier stage. To date, the Chinese Pharmacopoeia has not yet included a chapter on the NAT method for mycoplasma detection. In actual testing practice, we often refer to EP 2.6.7. The revisions of the new EP 2.6.7 have important reference and guiding significance for our actual mycoplasma detection practice.

Huzhou Shenke Biotechnology Co., Ltd. (HZSKBIO^®) has developed a series of mycoplasma DNA extraction and detection kits as well as validation strains, which comply with the requirements of the new edition of the European Pharmacopoeia for mycoplasma detection and support product compliance release.