Brocchini Steve, Balan Sibu, Godwin Antony, Choi Ji-Won, Zloh Mire, Shaunak Sunil
Department of Pharmaceutics, The School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, UK.
Nat Protoc. 2006;1(5):2241-52. doi: 10.1038/nprot.2006.346.
PEGylation has turned proteins into important new biopharmaceuticals. The fundamental problems with the existing approaches to PEGylation are inefficient conjugation and the formation of heterogeneous mixtures. This is because poly(ethylene glycol) (PEG) is usually conjugated to nucleophilic amine residues. Our PEGylation protocol solves these problems by exploiting the chemical reactivity of both of the sulfur atoms in the disulfide bond of many biologically relevant proteins. An accessible disulfide bond is mildly reduced to liberate the two cysteine sulfur atoms without disturbing the protein's tertiary structure. Site-specific PEGylation is achieved with a bis-thiol alkylating PEG reagent that sequentially undergoes conjugation to form a three-carbon bridge. The two sulfur atoms are re-linked with PEG selectively conjugated to the bridge. PEGylation of a protein can be completed in 24 h and purification of the PEG-protein conjugate in another 3 h. We have successfully applied this approach to PEGylation of cytokines, enzymes, antibody fragments and peptides, without destroying their tertiary structure or abolishing their biological activity.
聚乙二醇化已将蛋白质转变为重要的新型生物药物。现有聚乙二醇化方法的根本问题在于共轭效率低下以及形成异质混合物。这是因为聚（乙二醇）（PEG）通常与亲核胺残基共轭。我们的聚乙二醇化方案通过利用许多生物相关蛋白质二硫键中两个硫原子的化学反应性来解决这些问题。一个可接近的二硫键被温和还原以释放两个半胱氨酸硫原子，而不会干扰蛋白质的三级结构。通过双硫醇烷基化PEG试剂实现位点特异性聚乙二醇化，该试剂依次进行共轭以形成一个三碳桥。两个硫原子与选择性共轭到桥上的PEG重新连接。蛋白质的聚乙二醇化可在24小时内完成，聚乙二醇化蛋白质缀合物的纯化可在另外3小时内完成。我们已成功将此方法应用于细胞因子、酶、抗体片段和肽的聚乙二醇化，而不会破坏它们的三级结构或消除它们的生物活性。
