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.

Mammalian cell-produced therapeutic proteins: heterogeneity derived from protein degradation

“…Proteases of hybridoma cultures have been characterized as similar to lysosomal cathepsin D [58] or as a serine protease [59]. A similar finding was made in CHO cells expressing a MIMETIBODY TM protein [36 ] or expressing an Fc fusion protein [12 ]. For BHK cell cultures, a dipeptidyl-aminopeptidase has been described by Gawlitzek et al [60].…”

“…Terminal sialylation-deficient glycoproteins are rapidly cleared from blood, as stated previously. Enhanced structural heterogeneity of glycoproteins during the course of a batch culture was also found to be because of peptide degradation in the later part of the culture showing the most pronounced modulation [36 ]. The high viability of the cell population throughout the culture, and the period in which peptide degradation occurred, provided evidence for the secretion of proteases from metabolically active cells.…”

“…In fact, this strategy has been employed to design longer-lasting Aranesp [41], Enbrel [42], or GLP-1-MIMETIBODY TM [CNTO736/ CNTO3649, Refs. [11,36 ]] with extended circulating half-life. In each case, the product is engineered to contain one or more additional O-linked and/or N-linked glycosylation sites localized at the exposed domains of the glycoproteins.…”

“…Proteolytic clipping of a glucagon-like peptide1-Fc fusion protein [called GLP-1-MIMETI-BODY TM , Ref. [36 ]] as well as that of a recombinant Fc fusion protein has been described [12 ]. Several proteases such as serine proteases, elastase, collagenase, and plasminogen activator have been suspected to be secreted by CHO cells [37,38].…”

Proteomic Analysis of Bioreactor Cultures of an Antibody Expressing CHOGS Cell Line that Promotes High Productivity

“…'Global protein expression profiling using shotgun proteomics has been used in our laboratory to determine the protease profile of the CHO-GS host cell line [26] as well as gene expression profile of high producing cell lines [27]. Genome, transcriptome and proteome analysis of CHO cells have been reported multiple times [28][29][30][31][32][33].…”