Betaseron.

Betaseron, an analogue of human beta-interferon where serine was genetically engineered to substitute for cysteine at position 17, is produced in E. coli. The molecule is a small polypeptide of 165 amino acids with a single disulphide bond, and is non-glycosylated. The site-specific substitution was made to obtain a product that is more stable upon storage. Similar to native IFN-beta, Betaseron is hydrophobic in nature and has been shown to have the same panel of biological activities which includes antiviral activity against a variety of viruses, inhibition of cell growth, activation of natural killer cells, and binding to interferon receptors on the cell surface. Betaseron has been tested in a wide variety of clinical settings since 1983. The pivotal trial for the treatment of relapsing-remitting multiple sclerosis began in 1988. A PLA was filed for this indication in 1992 by Berlex and Chiron, and FDA approval was received in 1993. Betaseron is synthesized in E. coli and deposited as inclusion bodies. The manufacturing process involves solubilization and reduction of the insoluble protein, followed by purification by organic extraction, cystine oxidation and size exclusion chromatographic steps. The purified Betaseron is formulated with human serum albumin to maintain solubility at neutral pH. The complete primary sequence of Betaseron was verified by amino and carboxy-terminal sequence analysis, peptide mapping, amino acid analysis and fragment analysis after chemical cleavages. Overlapping amino acid sequence information confirmed that the amino acid sequence is the same as predicted by the DNA sequence. The amino-terminal methionine of Betaseron is removed after synthesis in E. coli. An intramolecular disulphide bond between Cys 31 and Cys 141 formed during the manufacturing process is routinely confirmed by peptide map analysis. The purity of Betaseron is assessed using a panel of analytical methods including non-reducing and reducing SDS-PAGE and reversed phase HPLC analysis where minor product-related components can be identified. These minor species were characterized with respect to their biological and biochemical properties, and identified using a variety of approaches including construction of additional, beta-interferon analogs. There is significant redundancy in the release testing of Betaseron. The amount of characterization information available on this relatively simple molecule along with the extensive manufacturing experience would suggest that some redundant testing could be eliminated for this well-characterized protein.