title: Use of toll-like receptor assays to detect and identify microbial contaminants in biological products.
abstract: Toll-like receptor (TLR)-expressing cells, for the first time, detected and identified a microbial contaminant in a product made in Escherichia coli using an old manufacturing process. It was suspected of having a microbial contaminant(s) because, although it tested negative by standard pyrogen assays, it was associated with adverse events in early clinical trials. The assay readout is the induction of NF-kappaB and/or cytokines in response to TLR activation. Four coded samples, labeled A to D, including a sample prepared by the older manufacturing process, were submitted. The cell lines were activated only by samples B and D. Sample D stimulated only Mono-Mac 6 and HEK-human TLR4 (hTLR4) cells and was later identified as lipopolysaccharide. Except for TLR3 cells, sample B stimulated cells bearing the different TLRs (TLRs 2, 4, 5, 7, 8, and 9) and nontransfected HEK293 cells. These data suggested that flagellin was the microbial contaminant, since TLR5, the receptor for flagellin, is known to be expressed constitutively on HEK293 cells. Moreover, purified flagellin from Salmonella enterica serovar Typhimurium behaved like sample B, stimulating HEK293 and HEK-hTLR5 cells but not HEK-hTLR3 cells, and this stimulation by flagellin and sample B was blocked by an anti-hTLR5 neutralizing antibody. Western blots showed bands positive for flagellin and sample B with the molecular sizes expected for the flagellins from S. Typhimurium and E. coli, respectively. Mass spectrometry data were consistent with the presence of flagellin in the manufacturer's sample B. Taken together, these data indicate that the microbial contaminant in sample B was flagellin and may have been associated with adverse events when the recombinant product was administered.
authors.affiliation: Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, HFM-345, Rockville, MD 20852, USA.
authors.forename: Li-Yun
authors.initials: LY
authors.lastname: Huang
authors.forename: James L
authors.initials: JL
authors.lastname: Dumontelle
authors.forename: Melissa
authors.initials: M
authors.lastname: Zolodz
authors.forename: Aparna
authors.initials: A
authors.lastname: Deora
authors.forename: Ned M
authors.initials: NM
authors.lastname: Mozier
authors.forename: Basil
authors.initials: B
authors.lastname: Golding
mesh_terms: D001688:Biological Products; D015153:Blotting, Western; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D004340:Drug Contamination; D004926:Escherichia coli / Q000276:immunology / Q000302:isolation & purification*; D005408:Flagellin / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008070:Lipopolysaccharides / Q000276:immunology / Q000302:isolation & purification; D013058:Mass Spectrometry; D016328:NF-kappa B / Q000378:metabolism; D012486:Salmonella typhimurium / Q000276:immunology / Q000302:isolation & purification*; D051193:Toll-Like Receptors / Q000276:immunology*
chemical_list: D001688:Biological Products; D016207:Cytokines; D008070:Lipopolysaccharides; D016328:NF-kappa B; D051193:Toll-Like Receptors; D005408:Flagellin