title: Characterization of the proteases involved in the N-terminal clipping of glucagon-like-peptide-1-antibody fusion proteins.
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.
authors.affiliation: Centocor Research and Development, Pharmaceutical Development Dept., 145 King of Prussia Road, Radnor, PA 19087, USA. hdorai@its.jnj.com
authors.forename: Haimanti
authors.initials: H
authors.lastname: Dorai
authors.forename: Aleixo
authors.initials: A
authors.lastname: Santiago
authors.forename: Marguerite
authors.initials: M
authors.lastname: Campbell
authors.forename: Q Mike
authors.initials: QM
authors.lastname: Tang
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Lewis
authors.forename: Yonghui
authors.initials: Y
authors.lastname: Wang
authors.forename: Qiao-Zhen
authors.initials: QZ
authors.lastname: Lu
authors.forename: Shiaw-Lin
authors.initials: SL
authors.lastname: Wu
authors.forename: William
authors.initials: W
authors.lastname: Hancock
mesh_terms: D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D052216:Glucagon-Like Peptide 1 / Q000737:chemistry*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry*; D040901:Proteomics; D011993:Recombinant Fusion Proteins / Q000737:chemistry*
chemical_list: D011993:Recombinant Fusion Proteins; D052216:Glucagon-Like Peptide 1; D010447:Peptide Hydrolases