Open Access Highly Accessed Research article

Reduction in C-terminal amidated species of recombinant monoclonal antibodies by genetic modification of CHO cells

Mihaela Škulj1*, Dejan Pezdirec1, Dominik Gaser1, Marko Kreft234 and Robert Zorec23

Author Affiliations

1 Sandoz Biopharmaceuticals, Mengeš, Lek Pharmacetucals d.d, Kolodvorska 27, 1234 Mengeš, Slovenia

2 Celica Biomedical Centre, Tehnološki Park 24, 1000 Ljubljana, Slovenia

3 Laboratory of Neuroendocrinology – Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia

4 Biotechnical Faculty, University of Ljubljana, Večna Pot 111, 1000 Ljubljana, Slovenia

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BMC Biotechnology 2014, 14:76  doi:10.1186/1472-6750-14-76

Published: 14 August 2014



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.


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.


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.

C-terminal amidation; Genetic modification; Recombinant monoclonal antibody