Enhancement Effect of a Variable Topology of a Membrane-Tethered Anti-Poly(ethylene glycol) Antibody on the Sensitivity for Quantifying PEG and PEGylated Molecules

Abstract

Sensitive quantification of the pharmacokinetics of poly(ethylene glycol) (PEG) and PEGylated molecules is critical for PEGylated drug development. Here, we developed a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for PEG by tethering an anti-PEG antibody (AGP3) via tethers with different dimensions on the surface of 293T cells (293T/S-αPEG, short-type cells; 293T/L-αPEG, long-type cells; 293T/SL-αPEG, hybrid-type cells) to improve the binding capacity and detection limit for free PEG and PEGylated molecules. The binding capacity of hybrid-type cells for PEG-like molecules (CH₃-PEG_5K-FITC (FITC = fluorescein isothiocyanate) and eight-arm PEG_20K-FITC) was at least 10-80-fold greater than that of 293T cells expressing anti-PEG antibodies with uniform tether lengths. The detection limit of free PEG (OH-PEG_3K-NH₂ and CH₃-PEG_5K-NH₂) and PEG-like molecule (CH₃-PEG_5K-FITC, CH₃-PEG_5K-SHPP, and CH₃-PEG_5K-NIR797) was14-137 ng mL^-1 in the hybrid-type cell-based sandwich ELISA. 293T/SL-αPEG cells also had significantly higher sensitivity for quantification of a PEGylated protein (PegIntron) and multiarm PEG macromolecules (eight-arm PEG_20K-NH₂ and eight-arm PEG_40K-NH₂) at 3.2, 16, and 16 ng mL^-1, respectively. Additionally, the overall binding capacity of 293T/SL-αPEG cells for PEGylated macromolecules was higher than that of 293T/S-αPEG or 293T/L-αPEG cells. Anchoring anti-PEG antibodies on cells via variable-length tethers for cell-based sandwich ELISA, therefore, provides a sensitive, high-capacity method for quantifying free PEG and PEGylated molecules.