Characterization of the proteases involved in the N-terminal clipping of glucagon-like-peptide-1-antibody fusion proteins

Haimanti Dorai*, Aleixo Santiago, Marguerite Campbell, Q. Mike Tang, Michael J. Lewis, Yonghui Wang, Qiao Zhen Lu, Shiaw Lin Wu, William Hancock

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    32 Citations (Scopus)

    Abstract

    In an attempt to develop high producing mammalian cell lines expressing glucagon-like-peptide-1-antibody fusion proteins (GLP-1), we have noted that the N-terminal GLP-1 portion of the fusion protein was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. The majority of the N-terminal clipped product appeared to be due to the removal of the entire biologically active peptide (30 amino acids) from the intact molecule. A number of parameters that influenced the degradative process were investigated. Additionally, protease inhibitors specific for each class of protease were tested. Results suggested that one or more serine-threonine class of protease(s) were involved in this process and inhibitors that are specific for this class of protease, including benzamidine hydrochloride could significantly inhibit the proteolytic degradation of the fusion proteins. Identification of the specific proteases involved in this process by shotgun proteomics methodology will pave the way for engineering the CHOK1SV cell line which will serve as a superior host for the production of future fusion protein products. American Institute of Chemical Engineers.

    Original languageEnglish
    Pages (from-to)220-231
    Number of pages12
    JournalBiotechnology Progress
    Volume27
    Issue number1
    DOIs
    Publication statusPublished - Jan 2011

    Keywords

    • Benzamidine hydrochloride
    • CHO cell line
    • GLP-1
    • Glycosylation
    • HTRA1
    • N-terminal clipping
    • Peptide-antibody fusion protein
    • Proteasome
    • Serine proteases
    • Shotgun proteomics

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