Secretion of Fc-amidated peptide fusion proteins by Chinese hamster ovary cells.

ABSTRACT: The therapeutic use of ?-amidated peptides (e.g. calcitonin, glucagon-like peptide) has increased dramatically, but there are major impediments to wider use of such peptides. Larger peptides are expensive to synthesize, and short plasma half-lives frequently limit the clinical circumstances in which the peptides would be useful. Both problems are potentially solved by producing peptides as fusions with the Fc region of human immunoglobulin.Glucagon-like peptide 1 (GLP1), peptide YY (PYY) and neuromedin U (NMU) were expressed and purified from stable CHO lines; since the ?-amide group is essential for full biological potency of many peptides, Fc-fusion peptides were expressed in CHO lines stably expressing the ?-amidating enzyme, peptidylglycine ?-amidating monooxygenase (PAM: EC 1.14.17.3). Purified fusion proteins were analyzed intact and after HRV3C rhinovirus protease cleavage, at a site in the linker separating the Fc region from the peptide, by mass spectrometry and amide-specific immunoassays.The Fc fusions were expressed at 1-2.5 ?g/mg cell protein and secreted at 5-20% of cell content per hour, in a peptide-specific manner. CHO cells express measurable endogenous PAM activity, amidating 25% of Fc-PYY and almost 90% of Fc-GLP1. Expression of exogenous PAM increased the level of peptide amidation to 50% of Fc-PYY and 95 % of Fc-NMU. The Fc-GLP1 fusions were 10,000-fold less active than synthetic GLP1 in a cell-receptor cyclic AMP-based assay, as expected since the amino terminal of GLP1 is essential for full biological activity. The Fc-PYY fusions were 100-fold less active than PYY-NH2 but 10-fold more active than non-amidated PYY-Gly.This type of approach can be used for the production of stabilized ?-amidated peptides aimed at clinical trials.

SUBMITTER: Carlson KR 

PROVIDER: S-EPMC4482046 | biostudies-other | 2015 Jun

REPOSITORIES: biostudies-other