Evaluation of different IRES-mediated tricistronic plasmid designs for expression of an anti-PCSK9 biosimilar monoclonal
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ORIGINAL RESEARCH PAPER
Evaluation of different IRES-mediated tricistronic plasmid designs for expression of an anti-PCSK9 biosimilar monoclonal antibody in CHO cells Thayana A. Cruz . Marcos B. Pinho . Leda R. Castilho
Received: 24 February 2020 / Accepted: 28 June 2020 Ó Springer Nature B.V. 2020
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 Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10529-020-02952-8) contains supplementary material, which is available to authorized users. T. A. Cruz M. B. Pinho L. R. Castilho (&) Chemical Engineering Program, Cell Culture Engineering Laboratory (LECC), COPPE, Federal University of Rio de Janeiro (UFRJ), Cx. Postal 68502, Rio de Janeiro, RJ 21941-972, Brazil e-mail: [email protected] T. A. Cruz L. R. Castilho Biochemistry Program, Federal University of Rio de Janeiro (UFRJ), IQ, Rio de Janeiro 21941-909, Brazil
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-LCIRES-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 antiPCSK9 mAb in stable CHO cell pools and provides benchmarks for fast production of different mAbs for characterization, formulation studies and pre-clinical investigation. Keywords Anti-PCSK9 monoclonal antibody Biosimilar development CHO cells Critical quality attributes IRES-mediated tricistronic plasmids
T. A. Cruz Libbs Farmaceˆutica, Sa˜o Paulo, SP 06807-461, Brazil Present Address: M. B. Pinho Caris Life Science, Inc., 4610 South 44th Place, Phoenix, AZ 85040, USA
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Biotechnol Lett
Introduction Monoclonal antibodies (mAbs) are the dominant class of biopharmaceuticals. Recombinant mammalian cell mAb production carried out in fed-batch mode can easily reach high titers ([ 1 g/L) in industrial scale (Kunert and Reinhart 2016). Recently, novel products for management of lipid disorders and cardiovascular risk have been
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