Design of homogeneous, monopegylated erythropoietin analogs with preserved in vitro bioactivity

Abstract

Objective-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 maleimidePEGs to engineered cysteine analogs of Epo, and to determine whether the PEGylated Epo cysteine analogs have improved pharmacokinetic properties in vivo. Materials and Methods-Thirty-fourEpo 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 kDamaleimide-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.Results-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.Conclusions-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.

Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin

“…Our data reveal that neighbor residue Thr27 is more suitable than Thr26 because cysteine substitution at this position does not affect N-glycosylation at position 24. As indicated in Table 3, the E89C analog has good criteria for cysteine PEGylation, high SAA and low RMSD, which is consistent with the previous study by Long et al 2 Even being PEGylated with a highmolecular-weight (20 kDa) PEG-maleimide, the E89C analog shows the same activity.…”

“…32 PEGylation of positions located in the N-terminal (R4 and L5) and C-terminal (R162, T163, and R166) in the EPO are not logical because of spatial restriction in interaction between EPO and its receptors. As shown by Long et al, 2 the attachment of 20 kDa PEG at positions A1, P3, and T163 decreases receptor binding affinity. In addition, it has been reported that terminal arginine of EPO is deleted from the CHO-expressed rhEPO.…”

“…In order for it to have therapeutic effect, it should be administered two to three times per week, which, in addition to the high cost of treatment, means frequent intravenous administration for patients. 2 Therefore, there has been growing interest in the development of long-acting products to be administered less often than existing products.…”

“…1 A defect in the production of EPO in the body leads to a decrease in red blood cells (anemia), which results from chronic kidney disease, chemotherapy, and azidothymidine treatment of HIV. 2 The production of recombinant human EPO (rhEPO) is developed genetically by many biopharmaceutical industries, as it is widely used in anemia disorders. The available rhEPO is a highly glycosylated protein (40% of molecular weight) and comprises 165 amino acids with a ∼35 kDa molecular weight.…”

“…These findings are consistent with previous studies that examined bioactivity of mutated EPOs by sitedirected mutagenesis. 2,28,29 In the third step, PEGylation factors must be assessed on stable modeled analogs. Factors affecting protein PEGylation include SAA, inherent nucleophilicity, and pKa of residue.…”