Differential sensitivity of lipegfilgrastim and pegfilgrastim to neutrophil elastase correlates with differences in clinical pharmacokinetic profile

To assess the basis of the different half‐lives of long‐acting human granulocyte colony‐stimulating factor (G‐CSF) drugs, the effect of neutrophil elastase on lipegfilgrastim and pegfilgrastim was investigated. Sensitivity to human neutrophil elastase (HNE) was evaluated by incubating the drugs with HNE followed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE). Drugs were also incubated with isolated human neutrophils followed by Western blot analysis. Lipegfilgrastim was more resistant to degradation with HNE or neutrophils than pegfilgrastim and appeared more intact on SDS‐PAGE gels and Western blots. Lipegfilgrastim retained more functional activity than pegfilgrastim after incubation with HNE (67% vs ∼9%, respectively) or neutrophils (80% vs ∼4%, respectively) as assessed in an NFS‐60 cell–based [3H]‐thymidine incorporation assay. The binding and affinity of untreated lipegfilgrastim and pegfilgrastim for G‐CSF receptors were evaluated using an NFS‐60 competitive G‐CSF receptor‐binding assay and surface plasmon resonance. Untreated drugs were also evaluated in the functional NFS‐60 thymidine incorporation assay. G‐CSF receptor binding, receptor affinity, and functional activity were comparable between untreated drugs. The results showed a greater resistance to neutrophil elastase degradation and concomitant retention of functional activity of lipegfilgrastim compared with pegfilgrastim, which potentially explains the clinical observations of a longer half‐life of lipegfilgrastim versus pegfilgrastim.