The following are key regulatory references:

USP <63>: Mycoplasma Tests

EP 2.6.7: Mycoplasmas

JP (17th Edition or latest): Mycoplasma Testing section

ICH Q5D: Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products

FDA CMC Biologics: Applicable to biologics, cell-based products, and vaccines, outlining detection requirements for mycoplasma

Yes. There are slight differences in the species required for validation, due to variations in traditional classification and growth behavior. The required fold increase for confirming effectiveness also varies—USP tends to be slightly stricter.
However, in practical application, these differences rarely result in significant outcome variations. Similar discrepancies exist in incubation temperature standards.
The number and type of mycoplasma strains required for detection also differ. The listed strains represent a minimum requirement. It is recommended to enhance internal SOPs by including additional species based on local epidemiological reports, industry standards, and operational experience.

The pharmacopoeias clearly state that the listed culture media are recommended, not mandatory. However, any alternative must be based on the same principles as the three-medium system.
The FDA inspection approach allows for deviations from USP or ICH if there is sufficient literature support and scientific rationale. These justifications must be logically sound and well-documented to gain acceptance during audits or hearings, which are often reviewed by domain experts.

Batch-to-batch validation is required even for well-defined bacterial media like TSA. This is due to strict quality control standards. Since mycoplasma media often contain natural components such as serum, variability between batches is more likely. As such, batch validation is even more critical for mycoplasma media than for bacterial ones.

Mycoplasma grows slowly. Although it can grow on agar, the growth rate is far slower than that of bacteria. In reality, only liquid media significantly promote proliferation. Therefore, the CFU obtained from agar can still serve as a valid indicator of the enrichment effect that occurred 10 days earlier in the liquid medium.

Regulatory updates must be based on rigorous experimental data and practical experience. Mycoplasma testing in biopharmaceuticals is relatively new, so regulatory agencies take a cautious approach and avoid adopting unproven methods prematurely.
In practice, except for Mycoplasma synoviae (which requires NAD), most other species can grow in all three media, though their growth rates and preferences vary. This reflects the limited research available on mycoplasma.
To ensure comprehensive detection of all strains listed in the regulations, the use of three different culture media remains standard—even if some newly developed single-medium formulations can support all strains. During FDA review, the three-medium system is still preferred.