Structure-Guided Engineering of a High-affinity anti-Methoxy Polyethylene Glycol Antibody for Sensitive Immunosensing of mPEGylated Therapeutics

Chiao-Yu Hsiao 1, Jun-Lun Meng 1, Jung-Zhe Hong 1, Meng-Hsuan Lin 1, Minh-Tram T. Nguyen1, Yu-Cheng Su*  

1Department of Biological Science and Technology, National Yang Ming Chiao Tung University

Sensitive quantification of methoxy poly(ethyleneglycol) (mPEG)-conjugated therapeutics for pharmacokinetic determination is critical for mPEGylated drug development. However, sensitive measurement of low-molecular-weight (lmw) mPEG compounds remains challenging due to epitope competition between backbone-specific anti-PEG antibodies. Here, we engineered a high-affinity methoxy-specific anti-mPEG antibody for sensitive quantification of free mPEG molecules and mPEGylated therapeutics. The affinity-enhanced h15-2Y antibody variant shows a 10.3-fold increase in mPEG-binding activity compared to parental h15-2b. h15-2Y-based sandwich ELISA can effectively quantify lmw mPEG5K and high-molecular-weight (hmw) mPEG20K at concentrations as low as 3.4 and 5.1 ng mL−1, respectively. Moreover, lmw mPEG compounds (560, 750, 1000, and 2000 Da) can be efficiently quantified via h15-2Y-based competitive ELISA with detection limits at nanomolar levels. This study provides a promising approach for application in the quantitative analysis of the various sizes of mPEG molecules to accelerate the timeline of mPEG-conjugated drug development.