Rational Development Of Lipegfilgrastim, a Novel Long-Acting Granulocyte Colony-Stimulating Factor, Using Glycopegylation Technology

Recombinant human granulocyte colony-stimulating factors (G-CSFs) are indicated for the prevention of chemotherapy-induced febrile neutropenia in patients receiving myelosuppressive chemotherapy. Filgrastim is a 175-amino-acid, short-acting, recombinant methionyl form of human G-CSF (r-metHuG-CSF) with a molecular weight of ∼19 kDa. The addition of polyethylene glycol (PEG) via PEGylation is a well-established technology that improves the physicochemical profile, enhances the solubility, and prolongs the half-life of therapeutic proteins. Pegfilgrastim, a PEGylated version of filgrastim, is currently the only available long-acting G-CSF, requiring fixed-dose subcutaneous (SC) administration only once per chemotherapy cycle. A new long-acting G-CSF, lipegfilgrastim, is under investigation for the prevention of chemotherapy-induced neutropenia. The rational design and development of lipegfilgrastim, and its novel manufacturing process, may provide a valuable fixed-dose, once-per-cycle, alternative long-acting G-CSF option. This analysis compares the design, protein chemistry, and physicochemical properties of lipegfilgrastim and pegfilgrastim, and evaluates the clinical implications of their differences.

The rational design of lipegfilgrastim focused on refining the PEGylation process. A novel technology platform was developed that enabled selective addition of PEG to a previously enzymatically attached glycan moiety instead of directly to the amino acid (as in standard PEGylation). Sequon scanning was used to scan the G-CSF protein to identify the best sites for glycosylation and glycoPEGylation that would have the least predicted effect on protein biologic activity. In recombinant G-CSF derived from Escherichia coli (E. coli), this was determined to be the single, natural, non-utilized O-glycosylation site. Addition of an O-glycan at this site was achieved by enzymatic activity of a selective, truncated N-acetylgalactosaminyltransferase isoform 2 fused with maltose-binding protein at the threonine residue within the chosen site. A 20-kDa PEG-sialic acid derivative was enzymatically transferred to the O-glycan with a sialyltransferase. Thus, the glycoPEGylation process modified only the O-glycosylation site of the G-CSF protein, resulting in a therapeutic product with an extended half-life.

The resultant glycoPEGylation product, lipegfilgrastim, is a long-acting, site-specific glycoPEGylated r-metHuG-CSF (molecular weight ∼38 kDa) produced by conjugation of a single 20-kDa PEG to the natural O-glycosylation site of G-CSF expressed in E. coli. In comparison, pegfilgrastim is produced by conjugation of a 20-kDa PEG to the N-terminal of G-CSF expressed in E. coli, and PEGylation results in a heterogeneous product with multiple PEGylated isoforms requiring further purification during the manufacturing process. Potency and receptor-binding studies have shown no apparent difference between lipegfilgrastim and pegfilgrastim (Scheckermann C, 2013, EHA, Abstract P1024). However, glycoPEGylation appears to provide lipegfilgrastim with different pharmacokinetic and pharmacodynamic profiles. In phase I studies in healthy volunteers, lipegfilgrastim 6 mg SC had ∼64% greater cumulative exposure and ∼36% higher peak exposure compared with pegfilgrastim 6 mg SC (Kohler E, 2012, MASCC/ISOO, Abstract A-445-0013-00997). Lipegfilgrastim also had a longer half-life compared with that of pegfilgrastim (geometric means, 32.4 hours vs 27.2 hours, respectively). In a double-blind, randomized, phase III trial, lipegfilgrastim was shown to be noninferior to pegfilgrastim in the duration of severe neutropenia in patients with breast cancer (Udo M, 2012, EHA, Abstract 1375).

GlycoPEGylation is an advanced and efficient process that permits the manufacture of a novel, customized, homogenous therapeutic protein, lipegfilgrastim, for the prevention of chemotherapy-induced neutropenia. The manufacturing process is simple, controllable, and scalable. Lipegfilgrastim has an extended half-life, permitting once-per-cycle SC administration and improved bioavailability and exposure and appears to be an effective alternative long-acting G-CSF.

Mahlert:Merckle Biotec GmbH (Teva Pharmaceuticals, Inc. Company): Employment. Schmidt:BioGeneriX GmbH (Teva Pharmaceuticals Inc. company): Employment. Allgaier:Merckle GmbH, Ulm, Germany (Teva Pharmaceuticals, Inc. company): Employment. Liu:Teva Pharmaceuticals, Inc: Employment. Müller:Teva Pharmaceuticals, Inc: Employment. Shen:Teva Pharmaceuticals, Inc: Employment.