Thayana A Cruz1,2,3, Marcos B Pinho1,4, Leda R Castilho5,6. 1. 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. 2. Biochemistry Program, Federal University of Rio de Janeiro (UFRJ), IQ, Rio de Janeiro, 21941-909, Brazil. 3. Libbs Farmacêutica, São Paulo, SP, 06807-461, Brazil. 4. Caris Life Science, Inc., 4610 South 44th Place, Phoenix, AZ, 85040, USA. 5. 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. leda@peq.coppe.ufrj.br. 6. Biochemistry Program, Federal University of Rio de Janeiro (UFRJ), IQ, Rio de Janeiro, 21941-909, Brazil. leda@peq.coppe.ufrj.br.
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.
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.