Polyethylene glycol (PEG) is a polyether, hydrophilic polymer derived from petroleum. PEG has a wide range of molecular weights and geometries and is found in everyday household items such as cleaners, laxatives, and cosmetics. Due to its ubiquitous presence in the environment, human exposure to PEG is frequent. PEG is commonly conjugated to therapeutic proteins to prolong the half-life and avoid aggregation (Mero et al., 2011). In addition, PEG is one of the major constituents of lipid-nanoparticles (LNP) used as drug delivery systems, e.g., in lipid nanoparticle messenger ribonucleic acid (LNP-mRNA), or in Accurin nanoparticles. PEG is a key component of the LNP steric barrier that helps protect the therapeutic payload from degradation thus prolonging its retention time in the body (Amoozgar and Yeo, 2012). PEG is generally recognized as safe (GRAS) by the Food and Drug Administration (FDA) (Yang and Lai, 2015).

It is well known that PEG can induce a humoral immune response in humans, as demonstrated by the high prevalence of circulating anti-PEG antibodies, and that the PEG moiety of biotherapeutics can also boost anti-PEG antibody levels (Ju et al., 2022). Anti-PEG antibodies may potentially impact drug safety and efficacy and consequently may need to be monitored in clinical drug development in applicable circumstances (Ju et al., 2023, Warren et al., 2021). Anti-PEG antibody assays may also be part of an immunogenicity testing paradigm for pegylated protein therapeutics or applied during the development of PEG-based delivery systems (e.g., LNP-mRNA) where justified. However, the development of fit-for-purpose human anti-PEG antibody assays has been a daunting task for bioanalytical scientists because of multiple challenges. The first challenge is the lack of assay specificity associated with commonly used LBA formats (e.g., the direct or indirect ligand binding assay), as these types of assay formats have been proven susceptible to interferences by non-specific serum proteins (Ehlinger et al., 2019). Moreover, the lack of specificity of such formats can be further compounded by the high prevalence of pre-existing anti-PEG antibodies. The second challenge is the lack of sensitivity in the detection of anti-PEG IgE antibodies. The bioanalytical field has made tremendous efforts in boosting the sensitivity of anti-PEG IgE antibody detection in order to better understand the relationship between PEGylated drug-associated anaphylaxis and specific IgE-mediated type 1 hypersensitivity reactions (HSR) (Bivi et al., 2020), (P. G. H. Gell RRAC., 1963). The third challenge is the source of critical assay reagents relevant to the detection of human anti-PEG antibodies. For example, the positive controls (PCs) in most anti-PEG antibody assays have been obtained from animals and thus not representative of anti-PEG antibodies in human serum (Krishna et al., 2015). In addition, the use of PCs from animals adds complexity when optimizing such assays, which typically utilize species-specific detector antibodies to bind the animal PC while anti-human antibody reagents are used to detect anti-PEG antibodies in the samples. Moreover, the conjugation of PEG (e.g., biotinylation and ruthenylation) has been challenging because conventional conjugation methods often lead to suboptimal incorporation of the conjugate molecule to PEG, which negatively impacts assay performance (Mero et al., 2011). Lastly, because multiple isotypes of anti-PEG antibodies can be induced, multiple assays to detect these isotype classes are often needed, which can increase blood volume requirements and consequently patient burden. Ideally, one multiplexed assay to detect and differentiate isotypes of anti-PEG antibodies is desired.

To address all these concerns, we developed a fit-for-purpose anti-PEG antibody assay with the end-goal of detecting anti-PEG IgM, IgG, and IgE antibodies using a multiplexed platform. Following a thorough literature review, we decided to develop such an assay by focusing on generating PEG conjugated reagents using a PEG molecular weight ranging from 2000 to 4000 Da. This range of PEG is currently used in LNP-mRNA based modalities for a wide range of disease indications and is informed by reports of the impact of anti-PEG antibodies on drug delivery and patient safety (Ju et al., 2022). With a final platform in mind, our initial focus was on the establishment of critical assay reagents using appropriate and innovative conjugation chemistry and sourcing of human anti-PEG IgM, IgG and IgE positive PCs (Bivi et al., 2020; Chen et al., 2016). Subsequently, we conducted intensive assay development and optimization to ensure the specificity, sensitivity, and robustness of isotype specific anti-PEG antibody detection in the multiplexed format, with details and results described in this manuscript.

Throughout this assay development and optimization, we proactively built our expertise in anticipation of supporting studies investigating PEG in the future.