Pegylated Versus Glycopegylated G-CSFs and Their Biochemical and Physiological Properties

Lipegfilgrastim and pegfilgrastim are long-acting, once-per-cycle recombinant granulocyte colony-stimulating factors (G-CSFs) developed to reduce the duration of neutropenia and the incidence of febrile neutropenia in cancer patients receiving chemotherapy (CTx). Lipegfilgrastim is recombinant methionyl human G-CSF that is modified at its natural O-glycosylation site (threonine¹³⁴) using a novel glycoPEGylation technology. Pegfilgrastim is a recombinant methionyl human G-CSF with a methoxy-polyethylene glycol-propionaldehyde covalently conjugated to its N-terminus. GlycoPEGylation generates functional PEGylated proteins with increased bioavailability and prolonged duration of action. Previous studies show that lipegfilgrastim has a 60% higher bioavailability and a 30% greater neutrophil response versus pegfilgrastim in healthy volunteers. When exposed to neutrophil elastase, G-CSF is rapidly cleaved and rendered inactive, suggesting that resistance to this degradation may result in an extended half-life.

The objectives of these studies were to characterize lipegfilgrastim versus pegfilgrastim with respect to bioavailability and affinity for the G-CSF receptor, to evaluate the effect of human neutrophil elastase on the degradation of lipegfilgrastim versus pegfilgrastim, and to evaluate the effect of degradation on the activity of these study drugs.

Lipegfilgrastim and pegfilgrastim binding to the G-CSF receptor was evaluated using: 1) a NFS-60-cell–based [¹²⁵I]-G-CSF competitive G-CSF receptor binding assay; and 2) a label-free surface plasmon resonance (SPR) technology by Biacore™ platform using a recombinant human G-CSF receptor. For the competitive binding assay, cocktails of NFS-60 cells, free [¹²⁵I]-G-CSF, and multiple concentrations of either (unlabeled) lipegfilgrastim or pegfilgrastim were incubated, and the cells were analyzed for bound [¹²⁵I]-G-CSF. Dose-dependent inhibition of [¹²⁵I]-G-CSF binding is indicative of the study drug binding to the G-CSF receptor. The SPR study evaluated binding kinetics and overall binding affinity between G-CSF receptor and lipegfilgrastim or pegfilgrastim. Degradation of lipegfilgrastim and pegfilgrastim by purified human neutrophil elastase was evaluated by incubation ± elastase and visualization using Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To evaluate the effect of elastase degradation on lipegfilgrastim or pegfilgrastim activity, samples treated with or without elastase for 15 or 120 minutes were incubated with NFS-60 cells for 20 hours, then pulsed with [³H]-thymidine for 4 hours. The level of active G-CSF–stimulated NFS-60 cell proliferation was measured by [³H]-thymidine incorporation.

In the cell-based [¹²⁵I]-G-CSF competitive G-CSF receptor binding assay, G-CSF receptor binding was equivalent between lipegfilgrastim and pegfilgrastim as indicated by inhibition of [¹²⁵I]-G-CSF binding (0.70±0.09 nM IC50 versus 0.72±0.18 nM IC50 (mean±SD). In the SPR study, the affinity for lipegfilgrastim and pegfilgrastim was 481±84 nM and 516±153 nM (mean±SD), respectively. Lipegfilgrastim treated with purified neutrophil elastase for 15 minutes appeared minimally degraded in SDS-PAGE gels and demonstrated a 67% relative activity in [³H]-thymidine proliferation assays compared with undigested lipegfilgrastim. In contrast, pegfilgrastim treated under the same conditions appeared markedly degraded and demonstrated an ∼9% relative activity compared with undigested pegfilgrastim. After treatment with purified neutrophil elastase for 120 minutes, lipegfilgrastim was noticeably degraded and the protein retained an ∼18% relative proliferative activity compared with undigested lipegfilgrastim, versus pegfilgrastim, which was almost entirely degraded and retained no activity compared with undigested pegfilgrastim.

Based on the present studies, there is no apparent difference in G-CSF receptor-binding affinity between lipegfilgrastim and pegfilgrastim. In addition, lipegfilgrastim showed a greater time-dependent resistance to neutrophil elastase degradation and a greater retention of functional activity, which may provide an explanation for the longer in vivo half-life of lipegfilgrastim versus pegfilgrastim.

Abdolzade-Bavil:Merckle GmbH (a Teva Pharmaceutical Inc., company): Employment. Cooksey:Teva Pharmaceuticals, Inc: Employment. Scheckermann:Merckle Biotec GmbH (a Teva Pharmaceuticals, Inc. company): Employment. Lammerich:Merckle/ratiopharm/Teva Pharm Industries: Employment. Pukac:Teva Pharmaceuticals, Inc: Employment. Krasney:Teva Pharmaceuticals, Inc: Employment. Allgaier:Merckle GmbH (a Teva Pharmaceuticals, Inc. company): Employment. Shen:Teva Pharmaceuticals, Inc: Employment. Müller:Teva Pharmaceuticals, Inc: Employment. Liu:Teva Pharmaceuticals, Inc: Employment. von Kerczek:2Teva Pharmaceuticals, Inc.: Employment.