ABSTRACT

During in vitro adventitious virus screening of a bulk harvest from a mammalian cell biopharmaceutical manufacturing process, the authors obtained results that at first suggested a viral contaminant, but that subsequently were found to represent a contamination with a nontuberculosis mycobacterium (Mycobacterium chelonae or M. abscessus).

Eukaryotic cell processes used in the manufacture of biologics are susceptible to contamination with adventitious agents. These agents include viruses, bacteria, fungi, mycoplasmas, and the so-called infectious proteins, such as transmissible spongiform encephalopathy agents. These entities may be introduced through contaminated raw materials (reagents and cell lines), during cell culture scale-up and fermentation, or during downstream processing. The methods used to conduct safety testing of biologics produced in cell cultures, and a survey of the types of contaminants that have been detected, have been described previously (1). The authors describe contamination of a mammalian cell-based biologic manufacturing process with a nontuberculosis mycobacterium (Mycobacterium chelonae or M. abscessus).

A bulk harvest from a mammalian cell bioreactor process was submitted to the authors for evaluation in a 28-day in vitro adventitious virus screening assay as part of an investigation of a bioreactor problem at a biologics manufacturing site. The bioreactor run had been terminated early, because of an abnormally high value for one of the in-process monitoring parameters. Microbial testing of the bioreactor contents, performed at the manufacturing site, had indicated an acceptable level of bioburden. As part of the investigation of the failed bioreactor run, the authors assayed the sample from the terminated bioreactor for potential viral contaminants according to the routine screening assay protocol. Eight days after inoculating the clarified bulk harvest material onto human (MRC–5), African green monkey (Vero), and Chinese hamster ovary (CHO–K1) detector cells, we observed floating clumps of an undetermined nature in the growth medium covering the monolayers. By 14 days post inoculation, the MRC–5 culture was devoid of viable cells. The Vero and CHO–K1 monolayers were intact and fully confluent at this time, though each displayed the floating clumps. Hemadsorption and hemagglutination testing of the various cultures, using chicken, guinea pig, and rhesus monkey erythrocytes, was completed on day 14 with negative results. The authors performed a passage of conditioned medium from each day-14 culture onto fresh cultures of detector cells. Eight days later, the MRC–5 monolayers displayed reduced confluency and abnormal cell morphology, including vacuolization. The Vero and CHO–K1 monolayers remained fully confluent at this time, although each displayed the floating clumps noted above. The hemadsorption and hemagglutination results were again negative. The MRC–5 portion of the study was repeated in its entirety from the beginning, and similar results were obtained.