Sensitive Quantification of PEGylated Compounds by Second-Generation Anti-Poly(ethylene glycol) Monoclonal Antibodies
Sensitive Quantification of PEGylated Compounds by Second-Generation Anti-Poly(ethylene glycol) Monoclonal Antibodies
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DOI：
10.1021/bc100067t
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发表时间：
2010-07-01
影响因子：
4.7
通讯作者：
Roffler, Steve R.
中科院分区：
文献类型：
--
作者：
Su, Yu-Cheng;Chen, Bing-Mae;Roffler, Steve R.
聚（乙二醇）（PEG）通常与化合物连接，以增加血清半衰期，降低免疫原性，并提高生物利用度。PEG结合物的准确和灵敏定量对于产品开发、药代动力学测量和功效研究至关重要。然而，由于PEG掩蔽表位，PEG化化合物可能难以定量。我们先前产生了两种针对PEG的单克隆抗体（AGP 3，IgM和E11，IgG），用于定量检测PEG化蛋白。我们现在报告两个第二代单克隆抗体的PEG（AGP 4，IgM和3.3，IgG），结合到PEG骨架的重复亚基，并促进更灵敏的定量范围更广的聚乙二醇化化合物的鉴定。其中使用AGP 4/3.3-生物素作为捕获/检测抗体的夹心ELISA允许PEG-Qdot 525的定量，其灵敏度比原始AGP 3/E11组合高14-50倍。Pegasys（PEG-干扰素α-2a）、PEG-Intron（PEG-干扰素α-2b）、Neulasta（PEG-G-CSF）和Lipo-Dox（PEG化脂质体阿霉素）也可以低ng/mL检测限定量。该测定耐受50%人血清或20%游离PEG分子的存在。这些新的抗PEG抗体似乎可用于多种PEG化化合物的定性和定量分析。
Poly(ethylene glycol) (PEG) is often attached to compounds to increase serum half-life, reduce immunogenicity, and enhance bioavailability. Accurate and sensitive quantification of PEG conjugates is critical for product development, pharmacokinetic measurements, and efficacy studies. However, PEGylated compounds can be difficult to quantify due to epitope masking by PEG. We previously generated two monoclonal antibodies to PEG (AGP3, IgM and E11, IgG) for quantitative detection of PEGylated proteins. We now report the identification of two second-generation mAbs to PEG (AGP4, IgM and 3.3, IgG) that bind to the repeating subunits of the PEG backbone and facilitate more sensitive quantification of a wider range of PEGylated compounds. A sandwich ELISA in which AGP4/3.3-biotin was employed as the capture/detection antibodies allowed quantification of PEG-Qdot 525 with 14-50-fold greater sensitivity than the original AGP3/E11 combination. Pegasys (PEG-interferon alpha-2a), PEG-Intron (PEG-interferon alpha-2b), Neulasta (PEG-G-CSF), and Lipo-Dox (PEGylated liposomal doxorubicin) could also be quantified with low ng/mL detection limits. The assay tolerated the presence of 50% human serum or 20% free PEG molecules. These new anti-PEG antibodies appear useful for qualitative and quantitative analysis of a wide range of PEGylated compounds.