Development of a PCR method for mycoplasma testing of Chinese hamster ovary cell cultures used in the manufacture of recombinant therapeutic proteins

Biological Enrichment of Mycoplasma Agents by Cocultivation with Permissive Cell Cultures

“…High Five insect cells (Invitrogen) were grown at 30°C Ϯ 1°C under an air atmosphere, using T75 flasks and Grace's insect cell culture medium (Grace's medium; Invitrogen) supplemented with 3.33 mg/liter lactalbumin hydrolysate, 3.33 mg/liter yeast hydrolysate, and 10% heat-inactivated FBS (Invitrogen). During the study, all working cell culture stocks were periodically tested for the absence of mycoplasmal, bacterial, and fungal contaminations by using light microscope examination of monolayers, fluorescence microscopy of Hoechst-stained cells, mycoplasma testing with a broth/agar culture method, and PCR amplification of the 16S rRNA gene (15) and the 16S-23S internal transcribed spacer (ITS) region (61). Using these methods, no mycoplasmal or any other bacterial contamination was ever detected in the working cell culture stocks.…”

“…Despite the reported ability of this mycoplasma testing procedure to efficiently detect all possible cell culture mycoplasmal contaminants, the overall testing procedure is time-consuming (a minimum of 28 days) and tedious and might not be suitable for biologics with shelf-lives that are shorter than the turnaround time for testing. In order to reduce the time required for mycoplasma testing, various approaches based on nucleic acid testing (NAT) technologies targeting different genetic markers of Mollicutes have been developed and proposed as potential alternatives to the current methods (15,31,33,54,56,57,63). However, although PCR methods have definite advantages over conventional microbiological methods in terms of analytical sensitivity, simplicity, and turnaround time required for testing, it continues to be unclear whether PCR-based methods can provide a limit of detection comparable or superior to those of conventional methods.…”

Contamination of Tissue Cultures by Mycoplasmas

“…For example, a PCR assay which applied readily available techniques in DNA extraction together with a modified single-step PCR using a primer pair that was homologous to a broad spectrum of mycoplasma species was proposed [97]. A high sensitivity and specificity for mycoplasma detection in cell production cultures was made possible through the combination of three key techniques: 8-methoxypsoralen and UV light treatment to decontaminate PCR reagents of DNA; hot-start Taq DNA polymerase to reduce nonspecific priming events; and touchdown PCR to increase sensitivity while also reducing nonspecific priming events.…”

Effect of storage conditions on detection of mycoplasma in biopharmaceutical products

“…Mammalian cell cultures used as substrates when producing biological products are required to be tested to verify the absence of a viable mycoplasma to ensure product purity and safety (Eldering et al 2004). Many test procedures have developed to detect mycoplasma, such as a classical culture assay, direct fluoreochromoe DNA staining, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR), which have advantages and disadvantages in terms of cost, time, reliability, specificity, and sensitivity (Uphoff et al 1992;Garner et al 2000).…”