Monoclonal tobacco cell lines with enhanced recombinant protein yields can be generated from heterogeneous cell suspension cultures by flow sorting.

Plant cell suspension cultures can be used for the production of recombinant pharmaceutical proteins, but their potential is limited by modest production levels that may be unstable over long culture periods, reflecting initial culture heterogeneity and subsequent genetic and epigenetic changes. We used flow sorting to generate highly productive monoclonal cell lines from a heterogeneous population of tobacco BY-2 cells expressing the human antibody M12 by selecting the co-expressed fluorescent marker protein DsRed located on the same T-DNA. Separation yielded ∼35% wells containing single protoplasts and ∼15% wells with monoclonal microcolonies that formed within 2 weeks. Thus, enriching the population of fluorescent cells from initially 24% to 90-96% in the six monoclonal lines resulted in an up to 13-fold increase in M12 production that remained stable for 10-12 months. This is the first straightforward procedure allowing the generation of monoclonal plant cell suspension cultures by flow sorting, greatly increasing the potential of plant cells as an economical platform for the manufacture of recombinant pharmaceutical proteins.

Comprehensive characterization of two different Nicotiana tabacum cell lines leads to doubled GFP and HA protein production by media optimization.

For over two decades, plant cell cultures have been a promising research platform to express recombinant and therapeutic proteins such as hormones, growth factors, full-size antibodies and antigens. Chosen as a good host for manufacturing recombinant proteins, the Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line has been studied in shake flasks by offline analysis of only a few growth parameters. The objective of this study is to comprehensively characterize the growth of a transgenic BY-2 cell line and to investigate the expression profile of the model protein GFP. Based on the correlations between nutrient consumption, cell growth and product formation, the intention is to improve the standard MS-medium. Hereby, multiple growth parameters were analyzed offline and online by using a respiration activity monitoring system (RAMOS). A reproducibly observed shift of the oxygen transfer rate (OTR) could be identified to indicate ammonium depletion in the medium. Concurrent with this ammonium depletion, the total protein concentration began to decrease. After the MS-medium was improved, the GFP concentration nearly doubled. When this improved ammonium enriched medium was applied to another transgenic tobacco cell line similar improvements to the amount of the glycoprotein influenza hemagglutinin (HA) produced by Nicotiana tabacum NT-1 cells could be achieved. Ultimately, this combined offline and online analysis can be successfully used for further cell line characterization and media optimization to improve growth and boost target product formation.