Mycoplasma contamination is a serious quality risk in cell-based manufacturing. These bacteria lack a cell wall and can grow in cell cultures without causing visible changes. A contaminated culture may continue to look normal while cell growth, metabolism, phenotype, and product quality change. Cell therapy analytical testing helps detect this risk as part of a wider quality control strategy.

For this reason, you should control mycoplasma at several points in the manufacturing process. Testing alone is not enough. Your control strategy should cover starting materials, cell banks, culture steps, equipment, facilities, in-process samples, and final product testing. FDA guidance also recommends testing at a stage where contamination is most likely to be detected, such as after culture pooling and before cell washing.

Where Mycoplasma Can Enter the Process?

Mycoplasma can enter a cell therapy process through several routes. FDA identifies animal-derived culture materials and the facility environment as important sources, especially when open culture systems are used.

Common risk points include:

• Donor-derived starting material
• Cell banks and working cell stocks
• Animal-derived materials and other raw materials
• Culture media and supplements
• Operators and facility activities
• Open handling steps
• Shared equipment
• Poorly controlled cleaning and transfer procedures

The risk can increase during cell expansion because cells remain in culture for a period of time. This gives a low-level contaminant an opportunity to multiply.

Mycoplasma can also pass through some filtration systems. FDA notes that Acholeplasma laidlawii can pass through a 0.2-micron filter but is retained by a 0.1-micron filter. This means you should not treat a standard 0.2-micron filtration step as proof that a process is free from mycoplasma.

Build Controls Around the Full Manufacturing Workflow

A strong control strategy starts before cells enter the production process.

1. Control Starting Materials and Cell Banks

Start with defined specifications for materials that come into contact with the cells. Your supplier qualification process should address microbiological quality and the risk from animal-derived materials.

Cell banks also need a documented testing strategy. The exact panel depends on the product and manufacturing process, but mycoplasma should be considered when cells are expanded or maintained in culture.

This is especially important for products made from cells that undergo repeated expansion or manipulation.

2. Control Cell Expansion

Cell expansion is one of the main areas where contamination can become established.

Your process should define:

• Culture conditions
• Maximum culture duration
• Permitted passage or expansion limits
• Sampling points
• In-process testing
• Acceptance criteria
• Actions for a suspected positive result

EMA guidance for genetically modified cell products specifically identifies mycoplasma as a biological parameter that can be used for in-process control. It also states that test methods and acceptance criteria should be defined.

For extended culture processes, in-process monitoring can provide an earlier warning than waiting until final product testing.

Use Testing at the Right Points

mycoplasma testing cell and gene therapy products should be designed around the product’s manufacturing process and release timeline. There is no single sampling point that fits every cell therapy.

FDA’s guidance for human somatic cell therapy recommends testing the product after cultures have been pooled for harvest but before cell washing. It also recommends testing cells and supernatant.

This sampling point has a practical reason. By harvest, material from multiple culture vessels may have been combined. Testing at this stage can provide information about the combined culture before further processing reduces the amount of material available for testing.

For short-lived cell products, conventional mycoplasma methods may not provide results quickly enough for release. FDA therefore describes PCR-based or other rapid methods as options during product development when traditional testing cannot fit the product’s dating period. The alternative method must have suitable sensitivity and specificity, supported by appropriate validation data.

Choose the Test Method Based on Risk

Traditional culture-based methods remain important reference methods. USP General Chapter <63> describes mycoplasma testing and notes that contamination may occur without turbidity or visible changes in the cells.

Nucleic acid amplification tests provide another approach. USP General Chapter <77>, published in the 2026 USP-NF, describes validation and application of nucleic acid amplification techniques for detecting mycoplasma contamination in biotechnology products and cell-based materials.

Each method has limits. Culture methods detect viable organisms but can require a long testing period. NAT methods can provide results much faster, but detection of nucleic acid does not by itself prove that the detected organism is viable. This distinction matters when you investigate a positive result.

Your cell therapy analytical testing program should therefore define the purpose of each method. A rapid method may support a time-sensitive release decision, while a compendial method may be used for confirmation, characterization, or other parts of the control strategy.

Strengthen GMP Manufacturing Controls

GMP manufacturing for cell therapy requires microbiological control throughout processing, not only at the testing stage.

Your facility controls should address personnel practices, cleaning, equipment, material movement, environmental controls, and open processing steps. Equipment and handling procedures should also be validated for microbiological control. EMA guidance specifically calls for validation of cell manipulation steps and equipment, including microbiological control and cleaning measures that prevent cross-contamination.

Environmental monitoring also needs careful thought. Routine bacterial and fungal monitoring does not automatically provide adequate mycoplasma control. FDA notes that mycoplasma environmental monitoring requires selective media.

You should also assess the risk from shared rooms and equipment. A contamination event in one process can affect another process when controls do not adequately separate materials, equipment, or personnel.

Manage Positive Results as a Process Failure

A positive mycoplasma result should trigger a documented investigation. Do not treat it as an isolated laboratory event.

Your investigation should examine:

1. The sample and test method
2. Laboratory controls and possible laboratory contamination
3. Raw materials and supplements
4. Cell banks and starting materials
5. Culture vessels and equipment
6. Facility and environmental data
7. Personnel activities
8. Previous batches and related products
9. Cleaning and disinfection records
10. Potential impact on affected material

The investigation should also determine whether other batches or materials may have been exposed to the same source.

Connect Testing With Process Knowledge

Mycoplasma testing cell and gene therapy products works best when testing is part of a wider contamination control strategy.

Do not rely on one final test to compensate for weak process controls. Your approach should combine qualified materials, controlled facilities, validated handling procedures, defined sampling points, suitable analytical methods, and documented responses to contamination.

The same principle applies to cell therapy analytical testing. Mycoplasma results should be considered alongside sterility, identity, viability, purity, potency, and other product-specific quality attributes. The testing program should reflect the risks created by your particular process.

For GMP manufacturing for cell therapy, the goal is to prevent contamination where possible and detect it early when prevention fails. FDA and EMA guidance support risk-based process design, defined in-process controls, process validation, and appropriate quality controls for cell and gene therapy products.

Conclusion

Mycoplasma control should follow the cells from starting material through expansion, harvest, processing, and release. Testing is one part of that system. Material controls, facility controls, aseptic handling, validated processes, and a clear investigation plan are equally important.

A well-designed program also considers the short shelf life of many cell products. Rapid methods may be needed when conventional testing cannot provide results before release, but their performance must be properly demonstrated.

For organizations developing cell-based products, Xellera Therapeutics can use this risk-based framework as a reference when building its contamination control and analytical strategy. The final approach should always match the product, process, applicable regulatory requirements, and supporting validation data.