Abstract
Objectives
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.
Results
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.
Conclusions
The expression platform proposed showed to be efficient to produce a high-quality anti-PCSK9 mAb in stable CHO cell pools and provides benchmarks for fast production of different mAbs for characterization, formulation studies and pre-clinical investigation.
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Acknowledgements
The authors would like to acknowledge the Brazilian research funding agencies CAPES, CNPq, and FAPERJ for financial support. The authors also thank Augusto Vieira (UEMP/UFRJ) and Eduardo Matos (CEMBIO/UFRJ) for support in the peptide mapping assay.
Supporting information
Supplementary Table 1. List of the forward (F) and reverse (R) primer pairs used for Sanger sequencing and PCR analysis carried out to validate correct subcloning into pCI-neo vector.
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Cruz, T.A., Pinho, M.B. & Castilho, L.R. Evaluation of different IRES-mediated tricistronic plasmid designs for expression of an anti-PCSK9 biosimilar monoclonal antibody in CHO cells. Biotechnol Lett 42, 2511–2522 (2020). https://doi.org/10.1007/s10529-020-02952-8
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DOI: https://doi.org/10.1007/s10529-020-02952-8