zahra shahrokh

Recombinant erythropoietins are used extensively in the management of anemia associated with chronic kidney disease (CKD). All the currently available erythropoietins are synthesized in Chinese hamster ovary (CHO) cell lines resulting in differences in glycosylation compared with human serum erythropoietin. We report the molecular characterization and pharmacokinetic properties of epoetin delta (Dynepo®, Shire plc), the only erythropoietin produced in a human cell line. A variety of techniques have been used to characterize epoetin delta, including amino acid sequencing, peptide mapping with reverse-phase HPLC/mass spectrometry, oligosaccharide profiling and MALDI-TOF of released glycans. Sialic acid and N-glycolylneuraminic acid (Neu5Gc) residues were quantified, following labeling of the released glycans, by reverse-phase HPLC analysis with fluorescence detection. Epoetin delta was produced as a highly pure and stable protein with the full human primary amino acid sequence. Neu5Gc...

Aggregation and precipitation are major events in the handling and aging of most protein pharmaceuticals. We demonstrate the utility of fluorescence spectroscopy in determining protein conformation in precipitates using basic fibroblast growth factor (bFGF) as an example. Conversion of the native to the soluble denatured from by chaotropes was accompanied by an increase in tryptophan emission. The emission spectra of resuspended precipitates were as reproducible as the spectra of the soluble form. The sum of emission spectra of native soluble bFGF and denatured precipitated bFGF was superimposable on the spectrum of the unfractionated suspension, suggesting that quantitative analysis of denatured aggregates in turbid protein formulations is possible. The ratio of tryptophan to tyrosine emissions increased with increasing extent of denaturation both in solution and in suspension. For example, salting out by ammonium sulphate increased the fluorescence index (indicative of denaturation) which was reversible upon dissolution. In addition, aging (35 degrees C) of bFGF in the presence of sulphated ligands produced precipitates with native-like fluorescence index, in contrast to denatured precipitates formed without ligands.

Apo2L/TRAIL (Apo2 ligand or tumor necrosis factor (TNF)-related apoptosis-inducing ligand) was discovered by its sequence homology to TNF and CD95L (refs. 1,2). Recombinant soluble human Apo2L/TRAIL is a candidate for clinical investigation in cancer therapy because it ...

Copyright © 2010 Frederick Furness Publishing 16 A focus on enzyme replacement therapies (ERT) for lysosomal storage diseases has led Shire Human Genetic Therapies, Inc (Shire HGT) to develop products for treating Fabry disease, Hunter syndrome, and type 1 Gaucher disease. These products are administered intravenously (IV) and are effective in treating the somatic symptoms of the disease. Developing ERT for diseases involving the CNS is a challenge because IV administered enzyme does not adequately cross the bloodbrain barrier (BBB) at a level needed for therapeutic effect. Moreover, formulations that are suitable for CNS administration are limited, posing a major challenge in generating stable products of adequate concentration. By developing new methods and formulations to deliver enzymes to the CNS, Shire HGT is at the forefront of developing ERT for treating lysosomal storage diseases with CNS involvement. ERT for CNS symptoms in Hunter syndrome and Sanfilippo A syndrome are in ...

The degradation products of basic fibroblast growth factor (bFGF) were isolated by ion exchange HPLC (HP-IEC) and characterized. The predominant product at pH 5 was a succinimide in place of aspartate15 as determined by LC/MS, N-terminal sequencing, and susceptibility to degradation at pH > 6.5. The rate of appearance of the succinimidyl-bFGF at 22 degrees C was comparable to that reported for small peptides, consistent with a high flexibility predicted for asp15-gly. Tryptic mapping together with [3H]-methylation indicated that iso-aspartate was formed at the position of asp15. Size exclusion HPLC indicated the presence of intact and truncated dimers and trimers which associated through disulfide linkages. Two truncated monomer forms were found that co-eluted by HP-IEC; the cleavages were determined to be at asp28-pro and asp15-gly using LC/MS and N-terminal sequencing. These degradation products which occurred at sites that are away from receptor or heparin binding domains of bFGF remained bioactive in a cell proliferation assay.

La presente invention concerne, entre autres, des compositions et des procedes pour l'administration au SNC d'enzymes lysosomales pour le traitement efficace de maladies liees au stockage lysosomal. Dans certains modes de realisation, la presente invention comprend une formulation stable pour l'administration intrathecale directe au SNC comprenant une proteine arylsulfatase A (ASA), un sel, et un tensioactif de type polysorbate pour le traitement de la maladie leucodystrophie metachromatique.