USP <63> Mycoplasma Tests

USP General Chapter <63> Mycoplasma Tests establishes the harmonized compendial methods for detecting mycoplasma contamination. Because mycoplasmas (Mollicutes) lack a cell wall, they are resistant to many antibiotics (e.g., penicillin), cannot be removed by standard 0.2 µm sterilization filters, and are undetectable by standard optical microscopy. This makes specific testing critical for patient safety.

Harmonization Status: This chapter is harmonized under the Pharmacopoeial Discussion Group (PDG) and is scientifically equivalent to Ph. Eur. 2.6.7 and JP G3 when applied as written in each pharmacopoeia.

1. Scope of Testing Mycoplasma testing is required at multiple stages of the biomanufacturing process:

• Master and Working Cell Banks (MCB/WCB)
• Viral Seed Stocks
• Unprocessed Bulk Harvest (UPB) (Harvest prior to purification)
• Raw Materials of animal origin (e.g., serum, trypsin)

2. The harmonized standard requires two methods unless scientifically justified.

• Direct Culture Method: To detect cultivable strains.
• Indicator Cell Culture Method: To detect fastidious, non-cultivable strains (e.g., M. hyorhinis).

3. Method A: Agar and Broth Culture (Direct Assay)

• Principle: Inoculation of samples into liquid broth and onto solid agar plates to allow colony formation.
• Incubation: The test period is not less than 21 days (incubated aerobically and anaerobically).
• Subculturing: Broth cultures are subcultured onto agar plates at appropriate intervals (e.g., early and mid-incubation) to promote the growth of slow-growing organisms.
• Detection: Microscopic examination for characteristic "fried egg" colony morphology.

4. Method B: Indicator Cell Culture (Indirect Assay)

• Principle: Inoculation of samples onto a specific indicator cell line known to support mycoplasma growth.
• Cell Lines: While Vero cells are commonly used, the chapter permits other validated substrates (e.g., CV-1 or 3T6 cells) capable of supporting the growth of fastidious strains.
• Staining: After incubation (typically 3–5 days), cells are stained with a DNA-binding fluorochrome (e.g., Hoechst 33258).
• Detection: Positive contamination is identified by extranuclear fluorescence (cytoplasmic DNA staining) or specific patterns on the cell surface, distinct from the nuclear staining observed in clean cells.

5. Alternative Methods (NAT / PCR) Rapid Nucleic Acid Amplification Techniques (NAT), such as Real-Time PCR, may replace one or both compendial methods when fully validated for equivalence.

• Validation Requirement: The alternative method must be validated to be at least equivalent to the compendial culture method.
• Sensitivity Target: A typical validation target is an LOD of ≤10 CFU/mL (or genomic copy equivalent), consistent with harmonized expectations and Ph. Eur. 2.6.7.