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Secretion of Fc-amidated peptide fusion proteins by Chinese hamster ovary cells.
Carlson, Kristina R; Pomerantz, Steven C; Li, Jiali; Vafa, Omid; Naso, Michael; Strohl, William; Mains, Richard E; Eipper, Betty A.
Affiliation
  • Carlson KR; Department of Neuroscience, UCONN Health Center, 263 Farmington Avenue, Farmington, CT, 06030-3401, USA. kristinarcarlson@gmail.com.
  • Pomerantz SC; Biologics Research, Biotechnology Center of Excellence, Janssen Research & Development, LLC, Spring House, PA, 19477, USA. spomeran@its.jnj.com.
  • Li J; Biologics Research, Janssen Research & Development, San Diego, CA, 92121, USA. jli65@its.jnj.com.
  • Vafa O; Biologics Research, Biotechnology Center of Excellence, Janssen Research & Development, LLC, Spring House, PA, 19477, USA. ovafa@its.jnj.com.
  • Naso M; Biologics Research, Biotechnology Center of Excellence, Janssen Research & Development, LLC, Spring House, PA, 19477, USA. mnaso@its.jnj.com.
  • Strohl W; Biologics Research, Biotechnology Center of Excellence, Janssen Research & Development, LLC, Spring House, PA, 19477, USA. wstrohl@its.jnj.com.
  • Mains RE; Department of Neuroscience, UCONN Health Center, 263 Farmington Avenue, Farmington, CT, 06030-3401, USA. mains@uchc.edu.
  • Eipper BA; Department of Neuroscience, UCONN Health Center, 263 Farmington Avenue, Farmington, CT, 06030-3401, USA. eipper@uchc.edu.
BMC Biotechnol ; 15: 61, 2015 Jun 27.
Article in En | MEDLINE | ID: mdl-26116580
ABSTRACT

BACKGROUND:

The therapeutic use of α-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.

METHODS:

Glucagon-like peptide 1 (GLP1), peptide YY (PYY) and neuromedin U (NMU) were expressed and purified from stable CHO lines; since the α-amide group is essential for full biological potency of many peptides, Fc-fusion peptides were expressed in CHO lines stably expressing the α-amidating enzyme, peptidylglycine α-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.

RESULTS:

The Fc fusions were expressed at 1-2.5 µg/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.

CONCLUSIONS:

This type of approach can be used for the production of stabilized α-amidated peptides aimed at clinical trials.
Subject(s)

Full text: 1 Database: MEDLINE Main subject: Recombinant Fusion Proteins / Immunoglobulin Fc Fragments / Amides Limits: Animals / Humans Language: En Year: 2015 Type: Article

Full text: 1 Database: MEDLINE Main subject: Recombinant Fusion Proteins / Immunoglobulin Fc Fragments / Amides Limits: Animals / Humans Language: En Year: 2015 Type: Article