Long Dana L, Doherty Daniel H, Eisenberg Stephen P, Smith Darin J, Rosendahl Mary S, Christensen Kurt R, Edwards Dean P, Chlipala Elizabeth A, Cox George N
Bolder BioTechnology, Inc., Boulder, CO 80301, USA.
Exp Hematol. 2006 Jun;34(6):697-704. doi: 10.1016/j.exphem.2006.02.011.
Erythropoietin (Epo) bioactivity is significantly reduced by modification of lysine residues with amine-reactive reagents, which are the most commonly used reagents for attaching polyethylene glycols (PEGs) to proteins to improve protein half-life in vivo. The aims of this study were to determine whether Epo bioactivity can be preserved by targeting attachment of maleimide-PEGs to engineered cysteine analogs of Epo, and to determine whether the pegylated Epo cysteine analogs have improved pharmacokinetic properties in vivo.
Thirty-four Epo cysteine analogs were constructed by site-directed mutagenesis and expressed as secreted proteins in baculovirus-infected insect cells. Following purification, monopegylated derivatives of 12 cysteine analogs were prepared using 20-kDa maleimide-PEGs. In vitro biological activities of the proteins were measured in an Epo-dependent cell proliferation assay. Plasma levels of insect cell-expressed wild-type Epo (BV Epo) and a pegylated Epo cysteine analog were quantitated by ELISA following intravenous administration to rats.
Biological activities of 17 purified Epo cysteine analogs and 10 purified pegylated Epo cysteine analogs were comparable to that of BV Epo in the in vitro bioassay. The only pegylated cysteine analogs that displayed consistently reduced in vitro bioactivities were substitutions for lysine residues, PEG-K45C and PEG-K154C. The pegylated Epo cysteine analog had a slower initial distribution phase and a longer terminal half-life than BV Epo in rats, but the majority of both proteins were cleared rapidly from the circulation.
Targeted attachment of maleimide-PEGs to engineered Epo cysteine analogs permits rational design of monopegylated Epo analogs with minimal loss of in vitro biological activity. Insect cell-expressed Epo proteins are cleared rapidly from the circulation in rats, possibly due to improper glycosylation. Site-specific pegylation appears to improve the pharmacokinetic properties of Epo.
用胺反应试剂修饰赖氨酸残基会显著降低促红细胞生成素（Epo）的生物活性，而胺反应试剂是将聚乙二醇（PEG）连接到蛋白质上以提高蛋白质体内半衰期最常用的试剂。本研究的目的是确定通过将马来酰亚胺-PEG靶向连接到Epo的工程化半胱氨酸类似物上是否可以保留Epo的生物活性，并确定聚乙二醇化的Epo半胱氨酸类似物在体内是否具有改善的药代动力学特性。
通过定点诱变构建了34种Epo半胱氨酸类似物，并在杆状病毒感染的昆虫细胞中作为分泌蛋白表达。纯化后，使用20 kDa的马来酰亚胺-PEG制备了12种半胱氨酸类似物的单聚乙二醇化衍生物。在Epo依赖性细胞增殖试验中测量蛋白质的体外生物活性。给大鼠静脉注射后，通过ELISA定量昆虫细胞表达的野生型Epo（BV Epo）和聚乙二醇化的Epo半胱氨酸类似物的血浆水平。
在体外生物测定中，17种纯化的Epo半胱氨酸类似物和10种纯化的聚乙二醇化Epo半胱氨酸类似物的生物活性与BV Epo相当。唯一显示体外生物活性持续降低的聚乙二醇化半胱氨酸类似物是赖氨酸残基的替代物，即PEG-K45C和PEG-K154C。在大鼠中，聚乙二醇化的Epo半胱氨酸类似物的初始分布阶段比BV Epo慢，终末半衰期比BV Epo长，但两种蛋白质的大部分都从循环中迅速清除。
将马来酰亚胺-PEG靶向连接到工程化的Epo半胱氨酸类似物上允许合理设计单聚乙二醇化的Epo类似物，且体外生物活性损失最小。昆虫细胞表达的Epo蛋白在大鼠循环中迅速清除，可能是由于糖基化不当。位点特异性聚乙二醇化似乎改善了Epo 的药代动力学特性。
