A host-cell-related proteolytic activity was identified in a recombinant Fc-fusion protein production process. This report describes the strategy applied to characterize and isolate the enzyme responsible for this degradation by combining cell culture investigation and dedicated analytical tools. After isolation and sequencing of the clipped fragment generated in post-capture material, enzymatic activity was traced in different culture conditions, allowing identification of viable CHO cells as the source of protease. Inhibitors and pH screenings showed that the enzyme belongs to an aspartic protease family and is preferably active at acidic pH. The protease was isolated by purification on a pepstatin A column and characterized as a protein related to cathepsin D. An additional metallo-protease inhibited by EDTA was identified with an optimum activity at neutral pH. This study is an example of how quality and stability of therapeutic recombinant molecules are strongly influenced by cell culture parameters.

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A novel variant of recombinant human growth hormone (r-hGH), isolated from biopharmaceutical preparations produced in E. coli, was identified and characterised. This variant contains a nonreducible thioether bridge near the C terminus between Cys182 and Cys189 and was characterised using various analytical techniques. As previous work by Cunningham and Wells (1993) highlighted the involvement of several residues in this part of the sequence in the binding and affinity of the molecule to its receptor, the presence of this modified intramolecular link may have important implications with regard to the biological behaviour of the molecule. Furthermore, as the conversion of a disulfide into a thioether was previously reported for a therapeutic monoclonal antibody (Tous et al., 2005), this may imply that disulfide bridges located in this part of the molecule have a generic susceptibility to thioether formation. This in turn is relevant to the biopharmaceutical industry for monitoring the integrity of disulfide bridges near the protein C terminus. The present study exhibits a state of the art physicochemical investigation for the unequivocal elucidation of a novel structure involving peptide mapping with mass spectrometry and de novo peptide sequencing. Changes in the higher order structure of the molecule were highlighted by near UV circular dichroism and molecular modelling.