Pegvaliase is an enzyme substitution therapy developed to lower blood phenylalanine (Phe) in adults with phenylketonuria (PKU). In phase 3 clinical studies， pegvaliase substantially reduced mean blood Phe in adult subjects with PKU. The most common type of adverse event observed in the pegvaliase clinical program was hypersensitivity adverse events (HAEs)， which included occurrences of arthralgia， rash， and pruritis. The most clinically relevant HAEs were acute systemic hypersensitivity reactions consistent with anaphylaxis observed in 4.6% of phase 3 patients. HAEs were more commonly observed around the time of high circulating immune complex (CIC) levels and complement activation， and the majority of subjects that experienced acute systemic hypersensitivity events were able to continue treatment， which is atypical for a classical IgE-mediated anaphylactic event， but common for type III hypersensitivity reactions. To investigate the alternative mechanism of type III hypersensitivity， serum samples collected shortly after hypersensitivity events (in phase 2 and 3 studies) were tested for anti-pegvaliase IgE using custom radioallergosorbent test and/or ImmunoCAP? (ThermoFisher Scientific， Waltham， MA) assay methods. All subjects with acute systemic hypersensitivity that were tested for anti-pegvaliase IgE at or near the time of event with one or both assays tested negative for IgE. As presented here， an investigation using selected study samples with high anti-drug antibody (ADA) titers demonstrated that presence of IgM and/or IgG ADA can interfere with measurement of a human anti-pegvaliase IgE surrogate positive control. A depletion method was therefore developed using protein A- and G-conjugated Sepharose to remove interfering IgG and IgM in serum samples to low levels (<45?mg/dL) before IgE testing. A final 2× concentration step brought the IgE concentration in the depleted sample to approximately the same level of the starting serum. Phase 3 study samples with sufficient volume remaining that previously tested negative for anti-pegvaliase IgE were re-tested after depletion of IgG and IgM. All samples again tested negative， confirming the original test results. Taken together， the clinical presentation， temporal association of HAEs with CIC levels and complement activation， and lack of anti-pegvaliase IgE suggest pegvaliase-associated acute systemic hypersensitivity events were not IgE-mediated. Furthermore， we describe a universal method that is broadly applicable to enzyme therapies for detection of low concentrations of drug-specific IgE in the presence of high titer anti-drug antibodies of different isotypes.