Recombinant Albumin-Partnering Technology: Development Of Balugrastim, a Novel Long-Acting Granulocyte Colony-Stimulating Factor

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.

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.

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).

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.

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.