Development of a novel anti-PEG antibody assay enabling investigation of potential immunogenicity triggered by the PEG moiety of biotherapeutics

Abstract

Polyethylene glycol (PEG) has been widely used in household products from skin care and cosmetics, to baby wipes and cleaners, as well as protein biotherapeutics. More recently, PEG has become a critical component of lipid nanoparticles (e.g., LNP or Accurin platforms) used to deliver small molecule and ribonucleic acid (RNA) therapeutics. A potential concern with therapeutics containing PEG is that the PEG moiety can induce anti-PEG antibodies that may impact patient pharmacokinetics (PK), safety and/or efficacy of the drug ((Ju et al., 2023), (Warren et al., 2021)). The accurate assessment of anti-PEG antibodies may be needed and their relationship to clinical PK, safety and efficacy mandates reliable bioanalytical methods with suitable specificity and sensitivity with characterization at the isotype level. Here we report the development of a novel multiplexed anti-PEG antibody sandwich-immunoassay, using an electrochemiluminescence (ECL) format to simultaneously detect major immunoglobulin (Ig) isotypes (IgM, IgG and IgE) of anti-PEG antibodies in human serum. As the initial step in the platform application's proof-of-concept (POC), we demonstrated that this first-ever multiplexed anti-PEG isotyping assay is highly specific with suitable sensitivities across all major immunoglobulin isotypes. Our holistic approach first focused on establishing critical assay components required for the multiplex format, followed by implementation of a sample pre-treatment procedure to optimize specificity and sensitivity. We then addressed non-specific matrix interference in the presence of potentially confounding factors, such as the high prevalence of pre-existing anti-PEG antibodies in human serum. This approach resulted in a specific and sensitive multiplex assay. Based on preliminary evaluation, the estimated assay sensitivities met regulatory expectations for clinical ADA assays (≤ 100 ng/mL). Notably, the estimated assay sensitivity for IgE isotypes of anti-PEG antibodies was ≤25 ng/mL, as determined using an IgE isotype specific anti-PEG antibody positive control.