Monitoring proteolysis of recombinant human interferon-γ during batch culture of Chinese hamster ovary cells

Proteolytic cleavage of recombinant human interferon-? (IFN-?) expressed in Chinese hamster ovary (CHO) cells during batch fermentation has been monitored by mass spectrometric peptide mapping. IFN-? was purified from cell-free culture supernatant by immunoaffinity chromatography and cleaved by endoprotease Asp-N. Peptide fragments were resolved by reverse-phase HPLC and identified by a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and automated N-terminal peptide sequencing. Using this approach, a peptide was identified as the C-terminal fragment of the IFN-? polypeptide. Analysis of this peptide by MS indicated that the recombinant IFN-? polypeptide secreted by CHO cells was truncated by at least ten amino acids, initially at Gln133-Met134. No full length (143 amino acids) polypeptide molecules were observed at any stages of the fermentation. Additional proteolytic cleavages at basic amino acids N-terminal of Gln133 occurred during the later stages of the culture resulting in a heterogeneous IFN-? polypeptide population with 'ragged' C-termini.