315. Identification and Control of Bacterial Contamination in an Academic Good Manufacturing Practice Facility
Authors: Timothy P. O'Connor, Neil R. Hackett, Robert G. Pergolizzi, Dolan Sondhi, Julie L. Boyer, Stephen M. Kaminsky, Ronald G. Crystal
Journal: Molecular Therapy (2006)

An ongoing monitoring program is an essential component of operating a Good Manufacturing Practice (GMP) facility. To support manufacture of clinical grade viral gene transfer vectors, the Belfer Gene Therapy Core Facility has a Class 100,000 GMP facility with three production laboratories, in which all open operations of production are conducted in Class 100 biological safety cabinets. The routine monitoring program measures viable and non-viable particles weekly and temporal trending is assessed to identify emerging problems. In July 2005, a series of vector batches were found to be contaminated by slow growing bacteria, necessitating an extensive investigation. Among the questions posed were: Are our environmental monitoring protocols adequate for detecting contamination? What are the sources of contamination? Do our cleaning protocols/products remove it? Can our production methods be improved to reduce the risk of contamination? The routine viable particle testing uses tryptic soy agar plates and contact slides, which we suspected may not support growth of certain bacteria that could grow in the DMEM medium (with 10% serum) used to culture HEK 293 cells for vector production. We created a semi-solid medium by addition of agar to cell culture medium and found that multiple samples that tested negative by the routine method now showed bacterial growth, including an occasional production contaminant later identified as Methylobacterium organophilum. Monitoring was repeated using this improved medium and several locations yielded bacteria upon swabbing, particularly the insides of cell culture incubators and water from incubator humidifiers. To identify the species and possible sources, 16S rRNA genes were PCR-amplified from bacterial isolates and, based on the sequence of the PCR product, the bacterium was identified. The most common contaminants were Bacillis licheniformis, Paenibacillus favisporus and Pseudomonas marginalis. Only one typical human flora, Staphylococcus epidermidis, was identified, in the access corridor where approved personnel enter without gowning. The data suggest that environmental bacteria, brought in inadvertently on materials, were the major contamination source. A complete cleaning of incubators was initiated, including flushing the water reservoirs and humidity chambers with a sporicidal cleaner, followed by normal rotation detergents. A complete facility cleaning followed the incubator cleaning and then another environmental monitoring round. Cycles of cleaning and expanded environmental monitoring continued until no contamination was detectable, at which point production resumed. The production process since has been contamination-free. Expanded cleaning and monitoring has been added to the routine facility maintenance, and procedures have been added to increase wipe down of materials entering the facility and during production. Modifications in monitoring and production resulting from the investigation represent improvements in our overall GMP quality assurance program.