Mollicutes removal is most commonly accomplished by filtration with 0.1 µm rated membrane filters. This filtration step requires process validation to assure the 0.1 micron rated filter performs as desired. The influence of process parameters—viscosity, differential pressure, contact time, fluid volume filtered, and organism species —determines the retentivity of the filter.
The variability of these factors makes it necessary to perform process validation, including product and process parameter challenge tests. Studies have shown that 0.1 um rated membrane filters that were able to retain challenges of >log 7 per cm2 of Acholeplasma laidlawii were unable to reach the same level of retentivity with Mycoplasma orale or M. pneumoniae.
The retention capabilities of different filters varies greatly, in fact, some filter vendors only guarantee proper reduction levels at specific higher Bubble Point values. Adsorptive retention is effective with some membrane polymers (Figure), but can cause unnecessary fouling, i.e., premature blockage. Additionally, adsorptive retentivity is affected by a multitude of process parameters and is, therefore, fairly unreliable.
Process validation also has a specific importance with 0.1 µm rated filters, as there is no standardization of the retentivity of such filters, which means variability in the performance of different 0.1 µm rated filters. A Parenteral Drug Association Mycoplasma Task Force is working on a standard challenge test proposal that could provide recommendations for the end-user’s testing of available 0.1 um filters.
Such tests could be performed in a laboratory setting, but retention reliability has to be performed with the actual product under process conditions. This approach, already being utilized with 0.2 um rated and viral removal filters, will provide assurance that the chosen filter is an appropriate one for the specific application. Redundancy or pure reliance on filter manufacturer’s data will not suffice.
The change from animal-derived media to plant peptone-derived media caused a recurrence of mycoplasmal contamination in cell culture media. This resurgence has resulted in a problem for the biotech industry, which relies heavily on large volumes of cell culture media for mammalian culture processes. A single preventive action will probably not prove adequate to assure contaminant-free medium; a multistep approach is necessary. Such an approach must be embraced by the end-user, and the raw material supplier must share the burden and enhance assurance.
Current detection, inactivation, and removal methods, in conjunction with appropriate process validation are the best options to prevent mycoplasma contamination.