ALBERT

Description: Introduction Various strategies have been used to extend the half-life of therapeutic proteins, including genetic fusion with carrier proteins. One such carrier protein is human serum albumin (HSA), a benign protein with minimal intrinsic biologic activity that is naturally present in the circulation at a high concentration. It has a long half-life (≈19 days in humans) and is highly soluble. Recombinant HSA produced from yeast retains the beneficial stabilizing properties of HSA while minimizing the potential disadvantages of a serum-derived product. Balugrastim, a novel, long-acting recombinant protein composed of HSA and human granulocyte colony-stimulating factor (G-CSF), was developed for once-per-cycle subcutaneous (SC) administration to provide a novel option for the prevention of severe neutropenia in patients with cancer receiving myelosuppressive chemotherapy. The rational design of balugrastim, differences in its protein chemistry compared with pegfilgrastim, and the clinical and practical implications are presented here. Methods During the design phase of balugrastim, HSA was deemed an ideal candidate as a carrier protein because of its wide distribution in the body, long half-life, and low potential for affecting biological activity of G-CSF. A highly engineered proprietary yeast strain was chosen to achieve high levels of expression and quality. Balugrastim is manufactured using recombinant DNA technology in the yeast Saccharomyces cerevisiae in contrast to pegfilgrastim, which is a PEGylated form of a G-CSF expressed in the bacterium Escherichia coli and then modified by chemical conjugation to polyethylene glycol. Balugrastim was purified using a combination of ion exchange and affinity and chromatography techniques. For clinical testing, sensitive immunogenicity and serum concentration assays were developed for the product. Results The manufacturing process produces balugrastim, a 759-amino-acid monomeric protein with a molecular mass of ≈85 kDa. It is a single continuous polypeptide chain in which residues 1–585 correspond to HSA and residues 586–759 correspond to the amino acid sequence of human G-CSF, connected via a peptide bond. The purified protein is 〉95% pure as determined by N-terminal sequencing. The result is a highly homogeneous product. The manufacturing process is straightforward, requiring no reformulations, additional chemical modifications, or secondary manufacturing, and is a scalable, modular production system. Balugrastim has a pharmacodynamic profile comparable to that of pegfilgrastim. In a clinical trial comparing balugrastim with pegfilgrastim in patients with breast cancer, the half-life of balugrastim 40 mg SC administered once per cycle was 37.7 hours, maximum plasma concentration was 875 ng/mL, and mean area under the concentration–time curve over 144 hours was 60321 h•ng/mL, providing sustained activity in the therapeutic window and stable blood levels (Pukac, MASCC/ISOO, 2012). Corresponding values for pegfilgrastim 6 mg SC were 47.1 hours, 164 ng/mL, and 11554 h•ng/mL, respectively. In this study, and in a randomized phase III trial in patients with breast cancer, balugrastim was noninferior to pegfilgrastim, with a safety profile similar to that of pegfilgrastim and low incidence of immunogenicity (Gladkov, ASCO, 2011). Conclusions Albumin partnering is an established technology used to generate innovative, half-life extended products. This technology formed the basis for the rational design for balugrastim, a novel once-per-cycle G-CSF for the prevention of severe neutropenia in patients with cancer receiving myelosuppressive chemotherapy. The technology provides balugrastim with several advantages, including a consistent, high-quality product with low immunogenic potential and an extended half-life that permits once-per-chemotherapy cycle administration. The low viscosity of balugrastim permits small needle size (29 gauge). Balugrastim, developed as an alternative to pegfilgrastim, has been shown to be noninferior to pegfilgrastim in clinical trials. Disclosures: Avisar: Teva Pharmaceuticals, Inc: Employment. Pukac:Teva Pharmaceuticals, Inc: Employment. Adar:Teva Pharmaceuticals, Inc: Employment. Barash:Teva Pharmaceuticals, Inc: Employment. Clark:Teva Pharmaceuticals, Inc: Employment. Liu:Teva Pharmaceuticals, Inc: Employment. Bock:Teva Pharmaceuticals, Inc: Employment. Shen:Teva Pharmaceuticals, Inc: Employment.

Description: Introduction 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. Methods 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. Results 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). Conclusions 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. Disclosures: 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.

Description: Background 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. Objective 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. Methods 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. Results 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. Conclusions 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. Disclosures: 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.