RESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Medicamentos Biossimilares/farmacologia , Sítios Internos de Entrada Ribossomal/genética , Pró-Proteína Convertase 9/genética , Sequência de Aminoácidos/genética , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Células CHO , Cricetulus/genética , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Humanos , Sítios Internos de Entrada Ribossomal/efeitos dos fármacos , Plasmídeos/genética , Plasmídeos/farmacologia , Pró-Proteína Convertase 9/imunologia , Pró-Proteína Convertase 9/farmacologia , TransfecçãoRESUMO
High-throughput screening (HTS) systems have emerged as important tools to provide fast and low cost evaluation of several conditions at once since it requires small quantities of material and sample volumes. These characteristics are extremely valuable for experiments with large number of variables enabling the application of design of experiments (DoE) strategies or simple experimental planning approaches. Once, the capacity of HTS systems to mimic chromatographic purification steps was established, several studies were performed successfully including scale down purification. Here, we propose a method for studying different purification conditions that can be used for any recombinant protein, including complex and glycosylated proteins, using low binding filter microplates.
Assuntos
Proteínas Recombinantes/isolamento & purificação , Biotecnologia/métodos , Cromatografia de Afinidade/métodos , Ensaios de Triagem em Larga Escala/métodosRESUMO
The pressures to efficiently produce complex biopharmaceuticals at reduced costs are driving the development of novel techniques, such as in downstream processing with straight-through processing (STP). This method involves directly and sequentially purifying a particular target with minimal holding steps. This work developed and compared six different 3-step STP strategies, combining membrane adsorbers, monoliths, and resins, to purify a large, complex, and labile glycoprotein from Chinese hamster ovary cell culture supernatant. The best performing pathway was cation exchange chromatography to hydrophobic interaction chromatography to affinity chromatography with an overall product recovery of up to 88% across the process and significant clearance of DNA and protein impurities. This work establishes a platform and considerations for the development of STP of biopharmaceutical products and highlights its suitability for integration with single-use technologies and continuous production methods. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:931-940, 2017.