Evaluation of different IRES-mediated tricistronic plasmid designs for expression of an anti-PCSK9 biosimilar monoclonal antibody in CHO cells.

Biotechnology Letters
Thayana A CruzLeda R Castilho

Abstract

To compare different approaches for the expression of an anti-PCSK9 biosimilar monoclonal antibody (mAb) in CHO cells using IRES-mediated tricistronic plasmid vectors combining different signal peptides, IRES elements and selection markers. Transient transfection indicated a similar level of secreted mAb 48 h post-transfection for all constructs. However, transfections carried out with circular plasmids showed a higher expression than with linearized plasmids. After two months under selection pressure, only part of the transfected pools recovered. The cultures co-transfected using two antibiotics as selection markers for double selection did not recover. Growth, metabolism and mAb production profiles of the only part of the transfected pools recovered resulting stable pools were compared and the stable pool transfected with circular L1-LC-IRES-H7-HC-IRES-NEO plasmid was chosen for further studies, due to higher cell growth and mAb production. Critical quality attributes of the protein A-purified mAb such as purity, homogeneity, binding affinity to PCSK9, and amino acid sequence were assessed confirming the success of the approach adopted in this study. The expression platform proposed showed to be efficient to produce a high-qu...Continue Reading

References

Oct 23, 2009·Biotechnology Progress·Say Kong NgMiranda G S Yap
Jun 12, 2012·New Biotechnology·Francesca ZagariFlorian M Wurm
Jun 30, 2012·Nature Reviews. Drug Discovery·Steven A BerkowitzGraham B Jones
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Jan 10, 2018·Preparative Biochemistry & Biotechnology·Hadi BayatAzam Rahimpour

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Methods Mentioned

BETA
protein folding
transfection
phosphotransferase
PCR
membrane filtration
Size exclusion chromatography
transfections
atomic force microscopy
affinity
glycosylation

Software Mentioned

Biotools
Vector NTI®
LC Solutions

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