Fig. 5
From: Optimizing production of Fc-amidated peptides by Chinese hamster ovary cells
GLP1-amide enzyme immunoassay. a. Validation of GLP1-amide assay using synthetic peptides. The amidated peptide representing the C-terminal half of GLP1 diluted in parallel with the GLP1-NH2 standard, as did culture medium from all the CHO cell lines producing Fc-GLP1-Gly (not shown). Both the Gly-extended GLP1 and the des-Gly peptide (terminating in Arg) showed no cross-reactivity in the assay. b. CHO cells expressing AP-GLP1-Gly (no exogenous PAM) or AP-GLP1-Gly and PAM1 (PAM1) were plated onto 12-well plates and incubated with control medium or medium containing 50 μM BCS for 16 h; spent media and cells were harvested and subjected to Western blot analysis for Fc and EIA for GLP1-NH2. The molar ratio of GLP1-NH2 to Fc is shown. c. Western blot for Fc from one of the experiments included in (c), demonstrating the lack of toxicity of the BCS treatment. d. Dose response for BCS, demonstrating the similar effect of BCS on the ability of endogenous CHO PAM and exogenous PAM1 to produce amidated GLP1-NH2; average of 2 assays