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

详细信息    查看全文

• 作者：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
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：December 20, 2016
• 年：2016
• 卷：88
• 期：24
• 页码：12371-12379
• 全文大小：569K
• ISSN：1520-6882

文摘

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 (mPEG_2K-NH₂ and mPEG_5K-NH₂) or PEG-conjugated small molecules (mPEG_5K-biotin and mPEG_5K-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.