Mycoplasma In-Process and Lot Release Testing: To PCR or Not to PCR

LEVEL: INTERMEDIATE M ycoplasma are the simplest self-replicating prokaryotes (1, 2). They infect a wide variety of eukaryotic hosts including humans, animals (birds, reptiles, fish, mammals), insects, plants, and bovine and ovine rumen. They are the smallest free-living prokaryotes, varying in size from 0.3 to 0.8 μm with a genome size of 580–2220 kb. They are contained by only a cell membrane without a rigid cell wall or peptidoglycan. Therefore, they are resistant to penicillin and can squeeze through 0.2-μm and 0.45-μm membranes routinely used for sterile filtration. They are frequent contaminants of cell cultures. Historically, about 15% of US cultures screened have been contaminated by mycoplasma. The contamination rate can be even higher in products from some other countries. Biologic products prepared using cell culture substrates are expected to be free of mycoplasma to assure safety, purity, potency, and consistency. Mycoplasma infection can affect nearly every cell culture parameter and result in decreased quantity or quality of product, inconsistency of manufacture, or possible adverse effects in recipients. Detection of mycoplasma is challenging because of their small size, limited turbidity produced in culture, no readily observable changes in growth or metabolic parameters with infection, requirements for enriched culture media or cell substrates for growth, and the wide diversity of mycoplasma species. Current testing requirements include US FDA codified regulations (21 CFR 610.30) (3), which apply to virus vaccines produced in cell cultures; however, some “noncultivable” organisms may not be detected with methods described in those requirements. The current industry standard for testing biologic products produced using cell substrates is described in Attachment 2 to the 1993 Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (4). This testing procedure applies to all biologics produced in cell substrates and includes a DNA staining procedure using indicator cell cultures to detect noncultivable strains, in addition to broth and agar culture methods. The culture methods of mycoplasma detection for cell bank and raw material release, in-process, and lot release testing, recommended in the documents mentioned above, have a long turnaround time (minimum 28 days). For most biological products, the mycoplasma culture test is the rate-limiting step for lot release. Alternative methods with shorter turnaround times, such as polymerase-chain-reaction (PCR)– based assays, have been developed recently. PCR methods are accepted for use as lot-release tests for biologics with extremely short shelf lives (shorter than the culture method turnaround time). But extending the use of PCR-based mycoplasma assays to other products raises concerns: namely, that PCR primers may not detect all possible mycoplasma species or that the methods may not be sufficiently sensitive.