title: Reduction in C-terminal amidated species of recombinant monoclonal antibodies by genetic modification of CHO cells.
abstract: BACKGROUND
  During development of recombinant monoclonal antibodies in Chinese hamster ovary (CHO) cells, C-terminal amidated species are observed. C-terminal amidation is catalysed by peptidylglycine α-amidating monooxygenase (PAM), an enzyme known to be expressed in CHO cells. The significant variations between clones during clone selection, and the relatively high content of amidated species (up to 15%) in comparison to reference material (4%), led us to develop a cell line with reduced production of C-terminal amidated monoclonal antibodies using genetic manipulation.
  
  
  RESULTS
  Initial target validation was performed using the RNA interference approach against PAM, which resulted in a CHO cell line with C-terminal amidation decreased to 3%. Due to the transient effects of small-interfering RNAs, and possible stability problems using short-hairpin RNAs, we knocked-down the PAM gene using zinc finger nucleases. Plasmid DNA and mRNA for zinc finger nucleases were used to generate a PAM knock-out, which resulted in two CHO cell lines with C-terminal amidation decreased to 6%, in CHO Der2 and CHO Der3 cells.
  
  
  CONCLUSIONS
  Two genetically modified cell lines were generated using a zinc finger nuclease approach to decrease C-terminal amidation on recombinant monoclonal antibodies. These two cell lines now represent a pool from which the candidate clone with the highest comparability to the reference molecule can be selected, for production of high-quality and safe therapeutics.
authors.affiliation: Sandoz Biopharmaceuticals, Mengeš, Lek Pharmacetucals d,d, Kolodvorska 27, 1234 Mengeš, Slovenia. mihaela-1.skulj@sandoz.com.
authors.forename: Mihaela
authors.initials: M
authors.lastname: Škulj
authors.forename: Dejan
authors.initials: D
authors.lastname: Pezdirec
authors.forename: Dominik
authors.initials: D
authors.lastname: Gaser
authors.forename: Marko
authors.initials: M
authors.lastname: Kreft
authors.forename: Robert
authors.initials: R
authors.lastname: Zorec
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry; D016466:CHO Cells; D003412:Cricetulus; D005818:Genetic Engineering / Q000379:methods; D006899:Mixed Function Oxygenases / Q000235:genetics; D009097:Multienzyme Complexes / Q000235:genetics; D011499:Protein Processing, Post-Translational; D034622:RNA Interference; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase