Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures
| Autor: | David Treiber, Brian D. Follstad, Zhimei Du, Yuling Zhang, Pranhitha Reddy, Brad Dell, Carole Heath, Dina Fomina-Yadlin, Ramsey A. Saleem, Arvia E. Morris, Craig Zupke, Matthew Leith, Brent Grisim, John D. McCarter, Rebecca E. Mccoy, Tharmala Tharmalingam |
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| Rok vydání: | 2014 |
| Předmět: |
Cell cycle checkpoint
glycosylation Cell Clone (cell biology) Cell Culture Techniques Bioengineering CHO Cells Applied Microbiology and Biotechnology recombinant antibody production Bioreactors Cricetulus Cyclin-dependent kinase Cricetinae specific productivity medicine Animals Enzyme Inhibitors biology Cell growth Chinese hamster ovary cell Cell Cycle Checkpoints Articles Cell cycle Cyclin-Dependent Kinases Recombinant Proteins product quality Cell biology medicine.anatomical_structure Cell culture biology.protein Biotechnology |
| Zdroj: | Biotechnology and Bioengineering |
| ISSN: | 1097-0290 |
| Popis: | The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures. |
| Databáze: | OpenAIRE |
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