FDA Points to Consider Method

M-700 Procedural Summary

This test satisfies the FDA Points to Consider for the Mycoplasma testing of Cell Cultures and Biologicals derived from cell substrates. The procedure is recommended for each lot of product harvest concentrate and the master cell banks, working cell stocks and cell substrates used for the manufacturing of these products. This method consists of the Direct Culture method combined with the Indicator Cell Culture procedure. (GMP service)

Direct Culture Method:

  • The direct culture procedure utilizes Fortified Commercial (FC) medium in both broth and agar formulations.
  • A 10 mL sample aliquot is inoculated into 50 mL of FC broth supplemented with 20% horse serum.  The broth culture is incubated aerobically.
  • 0.1 mL of sample is inoculated directly onto each of four FC agar plates.
  • The agar plates are incubated under appropriate aerobic and microaerophilic conditions as recommended in the FDA PTC procedure.
  • 0.1 mL sample of the broth culture is subcultured onto each of four fresh FC agar plates on Days 3, 7 and 14 post set-up.
  • All agar plates are examined microscopically for mycoplasma at 7 day intervals.
  • The broth culture is observed for signs of mycoplasmal growth (i.e. turbidity and/or pH changes.
  • Two positive controls, M. pneumoniae and M. orale (≤ 100 CFU) and a negative control are processed with each batch of samples.

DNA Fluorochrome Staining Assay with Indicator Cell Line:

  • The indicator cell culture procedure requires the inoculation of the test article onto two indicator cell cultures (slide cultures of Vero cells).
  • 1 mL aliquots of sample are inoculated onto each of two slide cultures of mycoplasmal free Vero cells.
  • Cell cultures are incubated at 36°C± 1°C with 5% CO2 and 95% air for a period of 3 to 5 days.
  • Following incubation, each slide culture is fixed and stained with a DNA-binding fluorochrome (Hoechst 33258). The extranuclear fluorescence staining pattern is then interpreted with UV-fluorescent microscopy up to 600x magnification.
  • Two positive control cultures, M.hyorhinis and M. orale (≤ 100 CFU), and a negative control culture are processed with each sample batch as specified.
  • Advantages of this assay include enhancement of sensitivity and specificity by reducing background from genetically unstable cell lines, amplification of mycoplasma titers and detection of "non-cultivable" organisms.
  • As a GMP test, quality assurance reports are provided with the final report packages.
  • Total testing time is 28 days.
Shipping Instructions:

Per FDA Points to Consider May 1993 Attachment # 2, samples should generally be stored frozen at ≤ -60°C for 24 hours or longer. Bionique Testing Laboratories requires that samples for the M-700 Mycoplasma Test be shipped and received frozen with dry ice remaining upon receipt at our facility. A 15 to 25 mL sample aliquot should be frozen in a sterile 50 mL polypropylene centrifuge tube or a sterile 40 mL Nalgene PETG screw cap bottle. Samples should be cryopreservative free. In addition, FDA recommends submitting product harvest concentrate for mycoplasma testing prior to purification, filtration, clarification or inactivation when possible. Place the frozen sample inside a plastic bag with crush proof packaging. Sample should be shipped overnight in an insulated container with sufficient dry ice to keep frozen for a period of 48 hours.

Please view our general sample submission instructions page for details on how to submit your sample(s) online.



Download Sample Submission Forms
FOR INFORMATION, CALL
GMP Service
Total Testing Time: 28 days
Sample Requirements: Minimum Test Volume 15 mL

Specializing In

Real-Time PCR Testing

Bionique has optimized the MycoSEQ Real-Time PCR Detection Assay for mycoplasma testing.

PCR Testing Services Harmonized USP Testing