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

BMC Biotechnology

Mihaela Škulj³  Dejan Pezdirec³  Dominik Gaser³  Marko Kreft²  Robert Zorec¹ 

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

[2] Biotechnical Faculty, University of Ljubljana, Večna Pot 111, 1000 Ljubljana, Slovenia

[3] Sandoz Biopharmaceuticals, Mengeš, Lek Pharmacetucals d.d, Kolodvorska 27, 1234 Mengeš, Slovenia

关键词: Recombinant monoclonal antibody;    Genetic modification;    C-terminal amidation;   

Others  :  1084726
DOI  :  10.1186/1472-6750-14-76

 received in 2014-05-30, accepted in 2014-08-08,  发布年份 2014

【 摘 要 】

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.

Conclusion

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.

【 授权许可】

   
2014 Škulj et al.; licensee BioMed Central Ltd.

【 图 表 】

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