Use of Toll-Like Receptor Assays To Detect and Identify Microbial Contaminants in Biological Products

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-κB 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.Biological products, including cellular and acellular vaccines, cells used in gene therapy, and plasma-derived and recombinant proteins, can become contaminated with many different types of organisms, e.g., gram-positive and gram-negative bacteria, fungi, viruses, parasites, and their by-products, during manufacturing. When the product is administered to a patient, these microbial contaminants may cause unwanted side effects, such as by inducing inflammation due to the release of cytokines or by acting as adjuvants that can potentially enhance the immunogenicity of a therapeutic protein.Traditionally, biological products are tested during the manufacturing process and at the time of lot release by the in vivo rabbit pyrogen test (RPT) and by the in vitro bacteria endotoxin test, commonly referred to as the Limulus amebocyte lysate (LAL) test. The requirement for final container product testing is stated in the U.S. Code of Federal Regulations, Title 21 (2, 3). These tests are designed to detect lipopolysaccharide (LPS), which is a constituent of gram-negative bacteria, or endotoxin and rely on nonhuman systems to predict human responses. Plasma-derived products and acellular vaccines can be sterile filtered before they are filled, and therefore, intact microorganisms can be removed. However, other microbial constituents, such as those derived from cell walls and nucleic acids (DNA and RNA), can evade filters and still end up in the final product. Recombinant proteins made in Escherichia coli, yeasts, or cell lines may also contain trace levels of host impurities, such that the final product may contain microbial components. These constituents may be difficult to detect by traditional methods.Recent studies have revealed that microbial pathogens possess specific pathogen-associated molecular patterns (13). The host innate immune system recognizes these pathogen-associated molecular patterns by using germ line-encoded pattern recognition receptors to elicit immune responses. Toll-like receptors (TLRs) are well-known pattern recognition receptors. Cells of the innate immune system utilize TLRs to detect cell wall components of bacteria, mycoplasma, fungi, and protozoa at the cell surface, whereas bacterial and viral nucleic acids are recognized by TLRs in a specialized intracellular endosomal compartment (12, 15, 26).Recent efforts have focused on the development of an in vitro test system that combines the sensitivity of the LAL test with the wide range of pyrogens detectable by the in vivo RPT. The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) evaluated the validation status of five in vitro test methods for assessment of the potential pyrogenicity of pharmaceuticals and other products proposed as potential replacements for the in vivo RPT (10). The ICCVAM proposed in vitro pyrogen tests that use enzyme-linked immunosorbent assays (ELISAs) for interleukin-1β (IL-1β) or IL-6 to measure increased levels of cytokine release when human blood cells, i.e., whole blood (WB), isolated monocytes, and a Mono-Mac 6 (MM6) cell line, are stimulated by endotoxin. On the basis of the findings of two published international validation studies, these five proposed in vitro pyrogenicity test methods are WB/IL-1β, WB/IL-6, peripheral blood mononuclear cell/IL-6, MM6/IL-6, and cryopreserved WB/IL-1β (4, 9, 11, 19).Armed with the knowledge of the critical role of TLRs in microbial detection as well as the need to avoid such issues as donor variability and the hazards associated with human blood products, our approach was to develop an assay that utilized cell lines expressing different TLRs. This was accomplished by using a panel of HEK293 cells transfected with different human TLRs. Initially, a screening test is performed by using the MM6 cell line. Previously, it was shown that MM6 cells respond to ligands to TLR2 and TLR4 (27). On the basis of the data presented in that paper, MM6 cells also respond to the TLR5 ligand flagellin but not to the TLR3, TLR7, TLR8, or TLR9 ligand. If a product sample tests positive with MM6 cells, then cell lines with more restricted TLR expression are used as detector cells to characterize the microbial ligand and, in turn, its microbial origin.Here we show the utility of this approach for the detection of a microbial contaminant in a sample obtained from a process used to make a recombinant product that had passed the standard lot release testing but that was associated with adverse events in humans. Not only was the panel of TLR-bearing cell lines able to detect a strong proinflammatory signal in the product, but also its use facilitated the identification of the microbial constituent at the molecular level.