Characterization of a HIR-Fab-IDS, Novel Iduronate 2-Sulfatase Fusion Protein for the Treatment of Neuropathic Mucopolysaccharidosis Type II (Hunter Syndrome).

BACKGROUND: Mucopolysaccharidosis type II is a severe lysosomal storage disease caused by deficient activity of the enzyme iduronate-2-sulfatase. The only medicinal product approved by the US Food and Drug Administration for enzyme replacement therapy, recombinant iduronate-2-sulfatase (idursulfase, Elaprase®), is a large molecule that is not able to cross the blood-brain barrier and neutralize progressive damage of the central nervous system caused by the accumulation of glycosaminoglycans. Novel chimeric protein HIR-Fab-IDS is an anti-human insulin receptor Fab fragment fused to recombinant modified iduronate-2-sulfatase. This modification provides a highly selective interaction with the human insulin receptor, which leads to the HIR-Fab-IDS crossing the blood-brain barrier owing to internalization of the hybrid molecule by transcytosis into endothelial cells adjacent to the nervous system by the principle of a 'molecular Trojan horse'. OBJECTIVES: In this work, the physicochemical and biological characterization of a blood-brain barrier-penetrating fusion protein, HIR-Fab-IDS, is carried out. HIR-Fab-IDS consists of an anti-human insulin receptor Fab fragment fused to recombinant iduronate-2-sulfatase. METHODS: Comprehensive analytical characterization utilizing modern techniques (including surface plasmon resonance and mass spectrometry) was performed using preclinical and clinical batches of HIR-Fab-IDS. Critical quality parameters that determine the therapeutic effect of iduronate-2-sulfatase, as well as IDS enzymatic activity and in vitro cell uptake activity were evaluated in comparison with the marketed IDS product Elaprase® (IDS RP). In vivo efficiency of HIR-Fab-IDS in reversing mucopolysaccharidosis type II pathology in IDS-deficient mice was also investigated. The affinity of the chimeric molecule for the INSR was also determined by both an enzyme-linked immunosorbent assay and surface plasmon resonance. We also compared the distribution of 125I-radiolabeled HIR-Fab-IDS and IDS RP in the tissues and brain of cynomolgus monkeys after intravenous administration. RESULTS: The HIR-Fab-IDS primary structure investigation showed no significant post-translational modifications that could affect IDS activity, except for the formylglycine content, which was significantly higher for HIR-Fab-IDS compared with that for IDS RP (~ 76.5 vs ~ 67.7%). Because of this fact, the specific enzyme activity of HIR-Fab-IDS was slightly higher than that of IDS RP (~ 2.73 × 106 U/µmol vs ~ 2.16 × 106 U/µmol). However, differences were found in the glycosylation patterns of the compared IDS products, causing a minor reduced in vitro cellular uptake of HIR-Fab-IDS by mucopolysaccharidosis type II fibroblasts compared with IDS RP (half-maximal effective concentration ~ 26.0 vs ~ 23.0 nM). The efficacy of HIR-Fab-IDS in IDS-deficient mice has demonstrated a statistically significant reduction in the level of glycosaminoglycans in the urine and tissues of the main organs to the level of healthy animals. The HIR-Fab-IDS has revealed high in vitro affinity for human and monkey insulin receptors, and the radioactively labeled product has been shown to penetrate to all parts of the brain and peripheral tissues after intravenous administration to cynomolgus monkeys. CONCLUSIONS: These findings indicate that HIR-Fab-IDS, a novel iduronate-2-sulfatase fusion protein, is a promising candidate for the treatment of central nervous system manifestations in neurological mucopolysaccharidosis type II.

A development of high-throughput HPLC method for the polysorbate 80 quantitation in protein therapeutic products.

A quantitative analysis of polysorbate 80 is an essential part of a characterization of therapeutic protein products. There are many methods to perform such type of analysis, including spectrophotometry and HPLC. However, there is no high-throughput method with high sensitivity and accuracy. In our work, we optimized conditions of polysorbate 80 hydrolysis to reduce the sample preparation time and developed a new isocratic reversed phase HPLC method for the quantification of oleic acid, the principal product of the hydrolysis. The validation of developed method shows that it is characterized by the repeatability less than 2.7% with the LOD about 25 ng (125 ppb), LOQ about 100 ng (500 ppb) within the analytical range of 0.025 to 1.5 mg/ml polysorbate 80 concentrations, accuracy between 92and108% and high precision (coefficient of variation less than3.2%). The total time needed for the analysis was about 1 h. The method could be routinely used for the quality control during therapeutic proteins manufacturing.

PASylation technology improves recombinant interferon-ß1b solubility, stability, and biological activity.

Recombinant interferon-ß1b (IFN-ß1b) is an effective remedy against multiple sclerosis and other diseases. However, use of small polypeptide (molecular weight is around 18.5 kDa) is limited due to poor solubility, stability, and short half-life in systemic circulation. To solve this problem, we constructed two variants of PASylated IFN-ß1b, with PAS sequence at C- or N-terminus of IFN-ß1b. The PAS-modified proteins demonstrated 4-fold increase in hydrodynamic volume of the molecule combined with 2-fold increase of in vitro biological activity, as well as advanced stability and solubility of the protein in solution as opposed to unmodified IFN-ß1b. Our results demonstrate that PASylation has a positive impact on stability, solubility, and functional activity of IFN-ß1b and potentially might improve pharmacokinetic properties of the molecule as a therapeutic agent.