For example, TLR 4 recognizes lipopolysaccharide, TLR 9 is activated by DNA encoding unmethylated cytosine-phosphate guanine motifs, and TLR 3 responds to double-stranded RNA. Multiple studies show that ligands for these TLR can be used to increase the immunogenicity of foreign proteins and may facilitate the breaking of tolerance to self. Indeed, data from the field of vaccine adjuvants shows that in mice, addition of 25–100 mg of CpG ODN or 10–100 mg of LPS to a protein antigen potentiates the production of antigen-specific antibodies, T cell survival and Th1 differentiation to the protein. Several studies have further shown that low levels of peptidoglycans or TLR 7 agonists can synergize with TLR 3 or TLR 4 agonists to foster secretion of IL-12 and other cytokines by dendritic cells and increase the immunogenicity of vaccines. However, to date, there are no formal studies addressing the impact of combinations of very low level TLR agonists on the immunogenicity of therapeutic proteins. In this study we hypothesized that TLR-stimulating impurities, at levels close to the limit of detection by currently used analytical methods, may stimulate the immune system and impact the product in terms of its immunogenicity, particularly when more than one type of impurity is present. Using LPS and synthetic DNA strands containing CpG motifs as a model we determined that trace levels of TLR 4 and 9 agonists synergize to induce cellular activation with increased cytokine and Z-VAD-FMK moa chemokine expression and antibody production both in vitro and in vivo. In the vaccine field, the concept of immunostimulatory combinations is being actively explored and studies using combinations of TLR and NLR agonists have shown promise in efforts to overcome tolerance to tumor antigens. In therapeutic proteins, where immune responses are not desired, factors that can contribute to product immunogenicity such as the degree of pre-existing tolerance, post-translation modifications, glycosylation patterns, and the presence of aggregates are carefully monitored. The data above shows that process-related impurities, especially those of microbial origin, may also play a key role in the immunogenicity of therapeutic proteins. The effect of IIRMIs was dose dependent and synergistic as levels of CpG ODN and LPS that individually induced no or very low levels of cytokine release by murine splenocytes, elicited polyclonal B cell activation with increased antigen-specific immunoglobulin and pro-inflammatory/Th1 cytokine output as well as up-regulation of co-stimulatory molecules on the cell surface of antigen presenting cells. This synergistic effect was then confirmed in vivo, as studies showed that the combination of 10 ng of LPS and 500 ng of CpG ODN, which do not induce an immune response when present individually, were sufficient to promote the immunogenicity of proteins and contribute to a clinically relevant break in tolerance to self.