Optimization of an Anti-poly(ethylene glycol) (anti-PEG) Cell-Based Capture System To Quantify PEG and PEGylated Molecules

Wen Wei Lin, Yuan Chin Hsieh, Yi-An Cheng, Kuo-Hsiang Chuang, Chien Chiao Huang, Chih Hung Chuang, I. Ju Chen, Kai Wen Cheng, Yun-Chi Lu, Ta Chun Cheng, Yeng Tseng Wang, Steve R. Roffler, Tian Lu Cheng

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9 Citations (Scopus)

Abstract

Sensitive determination of the pharmacokinetics of PEGylated molecules can accelerate the process of drug development. Here, we combined different anti-PEG Fab expressing 293T cells as capture cells (293T/3.3, 293T/6.3, and 293T/15-2b cells) with four detective anti-PEG antibodies (3.3, 6.3, 7A4, or 15-2b) to optimize an anti-PEG cell-based sandwich ELISA. Then, we quantified free PEG (mPEG2K-NH2 and mPEG5K-NH2) or PEG-conjugated small molecules (mPEG5K-biotin and mPEG5K-NIR797), proteins (PegIntron and Pegasys), and nanoparticles (Liposomal-Doxorubicin and quantum-dots). The combination of 293T/15-2b cells and the 7A4 detection antibody was best sensitivity for free PEG, PEG-like molecules, and PEGylated proteins with detection at ng mL(-1) levels. On the other hand, 293T/3.3 cells combined with the 15-2b antibody had the highest sensitivity for quantifying Lipo-Dox at 2 ng mL(-1). All three types of anti-PEG cells combined with the 15-2b antibody had high sensitivity for quantum dot quantification down to 7 pM. These results suggest that the combination of 293T/15-2b cells and 7A4 detection antibody is the optimal pair for sensitive quantification of free PEG, PEG-like molecules, and PEGylated proteins, whereas the 293T/3.3 cells combined with 15-2b are more suitable for quantifying PEGylated nanoparticles. The optimized anti-PEG cell-based sandwich ELISA can provide a sensitive, precise, and convenient tool for the quantification of a range of PEGylated molecules.

Original languageEnglish
Pages (from-to)12371-12379
Number of pages9
JournalAnalytical Chemistry
Volume88
Issue number24
DOIs
Publication statusPublished - Dec 20 2016

Keywords

  • Journal Article

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    Lin, W. W., Hsieh, Y. C., Cheng, Y-A., Chuang, K-H., Huang, C. C., Chuang, C. H., Chen, I. J., Cheng, K. W., Lu, Y-C., Cheng, T. C., Wang, Y. T., Roffler, S. R., & Cheng, T. L. (2016). Optimization of an Anti-poly(ethylene glycol) (anti-PEG) Cell-Based Capture System To Quantify PEG and PEGylated Molecules. Analytical Chemistry, 88(24), 12371-12379. https://doi.org/10.1021/acs.analchem.6b03614