Optical in-line biosensor for long-term continuous glucose monitoring and control in cell culture

Monitoring important process variables such as glucose in real-time is a major goal of bioprocess engineering, because it allows process control, which is not only essential for product quality and yield, but also important for the documentation and understanding of the production process and therefore relates to risk management [1]. This thesis deals with the development, thorough characterization and application of a disposable, optical in-line biosensor for monitoring and control of glucose in suspension cell culture. The in-line sensor, developed in this thesis, utilizes a commercially available oxygen sensor, that is coated with a crosslinked glucose oxidase (GOD) enzyme layer. The sensitivity of the sensor was tuned by the addition of a hydrophilic perforated diffusion membrane, to customize the dynamic range in order to meet the desired specifications. The biosensor was modelled in order to gain crucial insights into the internal concentration profile of the enzyme deactivating by-product hydrogen peroxide. The one-dimensional biosensor model revealed that the turnover rate of the enzyme GOD plays a crucial role for the functional stability of the biosensor in combination with the internal hydrogen peroxide accumulation. This insight was utilized to optimize the glucose biosensor for long-term continuous glucose monitoring over typical cell culture durations. A comprehensive biosensor characterization was performed to study the applicability and limitations of the developed biosensor for cell culture. Hereby, it was demonstrated that the sensor is sterilisable with beta, gamma and UV irradiation and is only subject to minor cross sensitivity to oxygen in combination with a reference oxygen sensor. The presented optical biosensor provides information in real-time and was therefore used in combination with a reference oxygen sensor to control the glucose level continuously in CHO cell culture with an automated feeding systems. It was found that the sialylation of the hyperglycosylated erythropoietin analog Darbepoetin alfa, could be significantly increased through continuous glucose feeding by retaining a high glucose level during the production phase of the cell culture. Therefore, the developed biosensor provides a valuable tool for optimizing culture conditions in biotechnological applications

Continuous optical in-line glucose monitoring and control in CHO cultures contributes to enhanced metabolic efficiency while maintaining darbepoetin alfa product quality

Great efforts are directed towards improving productivity, consistency and quality of biopharmaceutical processes and products. One particular area is the development of new sensors for continuous monitoring of critical bioprocess parameters by using online or in-line monitoring systems. Recently, we developed a glucose biosensor applicable in single-use, in-line and long-term glucose monitoring in mammalian cell bioreactors. Now, we integrated this sensor in an automated glucose monitoring and feeding system capable of maintaining stable glucose levels, even at very low concentrations. We compared this fed-batch feedback system at both low (< 1 mM) and high (40 mM) glucose levels with traditional batch culture methods, focusing on glycosylation and glycation of the recombinant protein darbepoetin alfa (DPO) produced by a CHO cell line. We evaluated cell growth, metabolite and product concentration under different glucose feeding strategies and show that continuous feeding, even at low glucose levels, has no harmful effects on DPO quantity and quality. We conclude that our system is capable of tight glucose level control throughout extended bioprocesses and has the potential to improve performance where constant maintenance of glucose levels is critical. © 2021 The Authors. Biotechnology Journal published by Wiley-VCH Gmb