RhDNase I is a Well Characterized Protein

Abstract

rhDNase I (Pulmozyme, Dornase Alpha) is a glycoprotein composed of 260 amino acids with two disulphide bridges and two sites for N-linked glycosylation. The purity and potency of the enzyme was demonstrated by a variety of methods. Techniques available today for the characterization of proteins provide a wealth of information. The techniques that proved most useful in the characterization of rhDNase I include peptide mapping/mass spectrometry, cation-exchange chromatography to measure deamidation of asparagine-74, potency by hydrolysis of a methyl green: DNA complex, high pH anion-exchange chromatography to profile released oligosaccharides and isoelectric focusing to compare charge heterogeneity. As clinical trials progressed it was necessary to modify the production of the protein to accommodate increased clinical demands. During the course of the phase III trials the fermentation vessel used in the production process was increased from 1000 L to the current market scale of 12000 L. Additional modifications of the purification steps to utilize larger fluid volumes were also necessary. Utilizing the above analytical techniques we were able to demonstrate the comparability of rhDNase I produced at the two scales without additional double-blind placebo-controlled clinical trials. The ability to use state-of-the-art protein chemistry methods to assess the comparability of rhDNase I made at two process scales resulted in rapid, worldwide approval of rhDNase I for the treatment of cystic fibrosis.