Online monitoring of the cell-specific oxygen uptake rate with an in situ combi-sensor.

In a biotechnological process, standard monitored process variables are pH, partial oxygen pressure (pO2), and temperature. These process variables are important, but they do not give any information about the metabolic activity of the cell. The ISICOM is an in situ combi-sensor that is measuring the cell-specific oxygen uptake rate (qOUR) online. This variable allows a qualitative judgement of metabolic cell activity. The measuring principle of the ISICOM is based on a volume element enclosed into a small measuring chamber. Inside the measuring chamber, the pO2 and the scattered light is measured. Within a defined measuring interval, the chamber closes, and the oxygen supply for the cells is interrupted. The decreasing oxygen concentration is recorded by the pO2 optode. This measuring principle, known as the dynamic method, determines the oxygen uptake rate (OUR). Together with the scattered light signal, the cell concentration is estimated and the qOUR is available online. The design of the ISICOM is focused on functionality, sterility, long-term stability, and response time behavior so the sensor can be used in bioprocesses. With the ISICOM, measurement of online and in situ measurement of the OUR is possible. The OUR and qOUR online measurement of an animal cell batch cultivation is demonstrated, with maximum values of OUR = 2.5 mmol L-1 h-1 and a qOUR = 9.5 pmol cell-1 day-1. Information about limitation of the primary and secondary substrate is derived by the monitoring of the metabolic cell activity of bacteria and yeast cultivation processes. This sensor contributes to a higher process understanding by offering an online view on to the cell behavior. In the sense of process analytical technology (PAT), this important information is needed for bioprocesses to realize a knowledge base process control.

Polysialic acid production using Escherichia coli K1 in a disposable bag reactor.

Polysialic acid (polySia), consisting of α-(2,8)-linked N-acetylneuraminic acid monomers plays a crucial role in many biological processes. This study presents a novel process for the production of endogenous polySia using Escherichia coli K1 in a disposable bag reactor with wave-induced mixing. Disposable bag reactors provide easy and fast production in terms of regulatory requirements as GMP, flexibility, and can easily be adjusted to larger production capacities not only by scale up but also by parallelization. Due to the poor oxygen transfer rate compared to a stirred tank reactor, pure oxygen was added during the cultivation to avoid oxygen limitation. During the exponential growth phase the growth rate was 0.61 h-1. Investigation of stress-related product release from the cell surface showed no significant differences between the disposable bag reactor with wave-induced mixing and the stirred tank reactor. After batch cultivation a cell dry weight of 6.8 g L-1 and a polySia concentration of 245 mg L-1 were reached. The total protein concentration in the supernatant was 132 mg L-1. After efficient and time-saving downstream processing characterization of the final product showed a protein content of below 0.04 mgprotein/gpolySia and a maximal chain length of ∼90 degree of polymerization.

Sensors for disposable bioreactors.

Modern bioprocess monitoring demands sensors that provide on-line information about the process state. In particular, sensors for monitoring bioprocesses carried out in single-use bioreactors are needed because disposable systems are becoming increasingly important for biotechnological applications. Requirements for the sensors used in these single-use bioreactors are different than those used in classical reusable bioreactors. For example, long lifetime or resistance to steam and cleaning procedures are less crucial factors, while a requirement of sensors for disposable bioreactors is a cost that is reasonable on a per-use basis. Here, we present an overview of current and emerging sensors for single-use bioreactors, organized by the type of interface of the sensor systems to the bioreactor. A major focus is on non-invasive, in-situ sensors that are based on electromagnetic, semiconducting, optical, or ultrasonic measurements. In addition, new technologies like radio-frequency identification sensors or free-floating sensor spheres are presented. Notably, at this time there is no standard interface between single-use bioreactors and the sensors discussed here. In the future, manufacturers should address this shortcoming to promote single-use bioprocess monitoring and control.