In situ cell differentiation monitoring of Catharanthus roseus suspension culture processes by NIR spectroscopy.

Plant suspension culture is attracting interest as a promising platform to produce biological medicines due to the absence of virus, prions or DNA related to mammals during the production process. However, the heterogenic plant cell proliferation nature is particularly challenging for establishing industrial processes based on innovative approaches currently used, particularly in the animal cell culture industry. In this context, while Process Analytical Technology (PAT) tools have been used to monitor classical parameters such as biomass dry weight, its use in cells heterogeneity has received limited attention. Therefore, the feasibility of in situ monitoring of cell differentiation in plant cell suspensions employing NIR spectroscopy and chemometrics was investigated. Off-line measurements of cell heterogeneity in term of cell differentiation and in-line NIR spectra captured in 3 L bioreactor cultures were employed to generate calibration models. Then models were tested to estimate the population distribution of parenchyma, collenchyma and sclerenchyma cells during Catharanthus roseus suspension cultures. Results have proven in situ NIR spectroscopy as a capable PAT tool to monitor differentiated cells accurately and in real-time. These results are the starting point to follow-up PAT systems so that plant cell culture heterogeneity may be better understood and controlled in biopharmaceutical plant cell cultures.

Still stable after 11 years: A Catharanthus roseus Hairy root line maintains inducible expression of anthranilate synthase.

Hairy root cultures generated using Agrobacterium rhizogenes are an extensively investigated system for the overproduction of various secondary metabolite based pharmaceuticals and chemicals. This study demonstrated a transgenic Catharanthus roseus hairy root line carrying a feedback-insensitive anthranilate synthase (AS) maintained chemical and genetic stability for 11 years. The AS gene was originally inserted in the hairy root genome under the control of a glucocorticoid inducible promoter. After 11 years continuous maintenance of this hairy root line, genomic PCR of the ASA gene showed the presence of ASA gene in the genome. The mRNA level of AS was induced to 52-fold after feeding the inducer as compared to the uninduced control. The AS enzyme activity was 18.4 nmol/(min*mg) in the induced roots as compared to 2.1 nmol/(min*mg) in the control. In addition, the changes in terpenoid indole alkaloid concentrations after overexpressing AS were tracked over 11 years. The major alkaloid levels in induced and control roots at 11 years are comparable with the metabolite levels at 5 years. This study demonstrates the long term genetic and biochemical stability of hairy root lines, which has important implications for industrial scale applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:66-69, 2017.