Unwanted immune responses to therapeutic proteins can severely impact their safety and efficacy. Studies show that the presence of trace amounts of host cells and process-related impurities that stimulate pattern recognition receptors (PRR) can cause local inflammation and enhance product immunogenicity. Here we used purified PRR agonists as model impurities to assess the minimal level of individual innate immune response modulating impurities (IIRMIs) that could activate a local immune response. We show that levels of endotoxin as low as 10 pg (0.01 EU), 1 ng for polyinosinic: polycytidylic acid (PolyI: C), 100 ng for synthetic diacylated liopprotein, thiazoloquinolone compound, or muramyl dipeptide, 1 mg for flagellin or b-glucan, or 5 mg for CpG-oligodeoxynucleotide increased expression of genes linked to innate immune activation and inflammatory processes in the skin of rhesusmacaques. Furthermore, spiking studies using rasburicase as a model therapeutic showed that the levels of PRR agonists that induced detectable gene upregulation in the skin were associated with increased immunogenicity for rasburicase. This study underscores the need for testing multiple IIRMIs in biologics, strengthening the connection between the local mRNA induction in skin, innate immune activation, and antibody development in primates, and provides an indication of the levels of IIRMI in therapeutic products that could impact product immunogenicity. Published by Elsevier Inc. on behalf of the American Pharmacists Association.