Protein spherulites for sustained release of interferon: preparation, characterization and in vivo evaluation.

This study develops protein spherulite, a new form of protein aggregation, for sustained-release applications of recombinant human interferon α-2b (rhIFN). rhIFN spherulites were prepared with different pH solutions with different kinds and concentrations of zinc salts at various incubation temperatures. rhIFN spherulites produced under different systems were of different morphology properties and in vitro release characters. Size-exclusion high-performance liquid chromatography analysis has shown that no protein aggregates were generated during spherulite formation, and cytopathic inhibition assay has demonstrated that the spherulitic rhIFN well maintained its structure and antiviral activity. In vivo studies showed that rhIFN spherulites provided a significantly prolonged pharmacokinetics profile profile as compared with the soluble rhIFN formulation, and the relative bioavailability based on serum rhIFN levels was about 170%. The work described here demonstrates the possibility of protein spherulites as a long-acting formulation for rhIFN.

Fibrillation of carrier protein albebetin and its biologically active constructs. Multiple oligomeric intermediates and pathways.

We showed that the genetically engineered carrier-protein albebetin and its biologically active constructs with interferon-alpha(2) octapeptide LKEKKYSP or differentiation factor hexapeptide TGENHR are inherently highly amyloidogenic at physiological pH. The kinetics of fibrillation were monitored by thioflavine-T (ThT) binding and the morphological changes by atomic force microscopy. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils. Comparative height and volume microscopic measurements allowed us to identify two distinct types of oligomeric intermediates: pivotal oligomers ca. 1.2 nm in height comprised of 10-12 monomers and on-pathway amyloid-competent oligomers ca. 2 nm in height constituted of 26-30 molecules. The former assemble into chains and rings with "bead-on-string morphology", in which a "bead" corresponds to an individual oligomer. Once formed, the rings and chains remain in solution simultaneously with fibrils. The latter give rise to protofilaments and fibrils, and their formation is concomitant with an increasing level of ThT binding. The amyloid nature of filamentous structures was confirmed by a pronounced ThT and Congo red binding and beta-sheet-rich far-UV circular dichroism. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in amyloid assembly. Peptides, either fused to albebetin or added into solution, and an increased ionic strength promote fibrillation of albebetin (net charge of -12) by counterbalancing critical electrostatic repulsions. This finding demonstrates that the fibrillation of newly designed polypeptide-based products can produce multimeric amyloid species with a potentially "new" functionality, raising questions about their safety.

Interferon immunogenicity: technical evaluation of interferon-alpha 2a.

Observations from some studies with interferon-alpha 2a (IFN-alpha 2a) have shown the presence of neutralizing antibodies in a proportion of patients. As a result, an investigation into the production of antibodies to IFN-alpha 2a was undertaken. A number of technical aspects of its production and storage were investigated, including the possibility of an incorrect structure, which could affect the immunogenicity of the IFN-alpha 2a molecule. These investigations demonstrated the presence, in vials of IFN-alpha 2a, of both interferon-interferon (IFN-IFN) aggregates and aggregates of interferon with human serum albumin (HSA), the excipient of the galenical form of IFN-alpha 2a (IFN-HSA) aggregates. The amount of aggregates is temperature dependent, there being very little increase in aggregate content over time when vials are stored at 4 degrees C. The relative immunogenicity of IFN-alpha 2a increased when the vials were stored at ambient temperature but not when stored at 4 degrees C. These findings demonstrate that the immunogenicity of IFN-alpha 2a is likely to be related to the storage temperature. Storage of IFN-alpha 2a vials at 2-8 degrees C is now recommended. A new formulation has been introduced that does not contain HSA as an excipient, removing the possibility of IFN-HSA aggregation.

A homology model of human interferon alpha-2.

An atomic coordinate five alpha-helix three-dimensional model is presented for human interferon alpha-2 (HuIFN alpha 2). The HuIFN alpha 2 structure was constructed from murine interferon beta (MuIFN beta) by homology modeling using the STEREO and IMPACT programs. The HuIFN alpha 2 model is consistent with its known biochemical and biophysical properties including epitope mapping. Lysine residues predicted to be buried in the model were primarily unreactive with succinimidyl-7-amino-4-methylcoumarin-3-acetic acid (AMCA-NHS), a lysine modification agent, as shown by mass spectrometric analysis of tryptic digests. N-terminal sequence analysis of polypeptides generated by limited digestion of HuIFN alpha 2 with endoproteinase Lys-C demonstrated rapid cleavage at K31, which is consistent with the presence of this residue in a loop in the proposed HuIFN alpha 2 model. Based on this model structure potential receptor binding sites are identified.