In-situ imaging sensors for bioprocess monitoring: state of the art.

Over the last two decades, more and more applications of sophisticated sensor technology have been described in the literature on upstreaming and downstreaming for biotechnological processes (Middendorf et al. J Biotechnol 31:395-403, 1993; Lausch et al. J Chromatogr A 654:190-195, 1993; Scheper et al. Ann NY Acad Sci 506:431-445, 1987), in order to improve the quality and stability of these processes. Generally, biotechnological processes consist of complex three-phase systems--the cells (solid phase) are suspended in medium (liquid phase) and will be streamed by a gas phase. The chemical analysis of such processes has to observe all three phases. Furthermore, the bioanalytical processes used must monitor physical process values (e.g. temperature, shear force), chemical process values (e.g. pH), and biological process values (metabolic state of cell, morphology). In particular, for monitoring and estimation of relevant biological process variables, image-based inline sensors are used increasingly. Of special interest are sensors which can be installed in a bioreactor as sensor probes (e.g. pH probe). The cultivation medium is directly monitored in the process without any need for withdrawal of samples or bypassing. Important variables for the control of such processes are cell count, cell-size distribution (CSD), and the morphology of cells (Höpfner et al. Bioprocess Biosyst Eng 33:247-256, 2010). A major impetus for the development of these image-based techniques is the process analytical technology (PAT) initiative of the US Food and Drug Administration (FDA) (Scheper et al. Anal Chim Acta 163:111-118, 1984; Reardon and Scheper 1995; Schügerl et al. Trends Biotechnol 4:11-15, 1986). This contribution gives an overview of non-invasive, image-based, in-situ systems and their applications. The main focus is directed at the wide application area of in-situ microscopes. These inline image analysis systems enable the determination of indirect and direct cell variables in real time without sampling, but also have application potential in crystallization, material analysis, polymer research, and the petrochemical industry.

Development of a flow-through microscopic multitesting system for parallel monitoring of cell samples in biotechnological cultivation processes.

The automated monitoring of cell variables in cultivation processes is a key technology in modern bioscience. In this article an innovative analytical tool for monitoring of cell densities in shaking-flask cultivations--consisting of a flow-through microscope with an automated image analysis software integrated in a FIA sampling system--is presented. This atline multitesting system was optimized by varying the height of the microscopes sampling zone. A calibration of the system was performed by correlating the FIA result peaks to known concentrations of Baker's yeast. It was successfully applied in cell density monitoring of cultivation processes of Saccharomyces cerevisiae with a good correlation with offline methods, and further used to monitor 3 parallel cultivations. This methodology was successfully transferred to Bacillus megaterium cultures and applied to measure the cell densities of parallel cultivation setups of B. megaterium and S. cerevisiae.

Adsorption and separation of proteins by a smectitic clay mineral.

The adsorption of proteins by a smectitic clay mineral was investigated. The clay used in this study is a mixture of montmorillonite and amorphous SiO(2). Due to the high porosity the montmorillonite units are accessible for protein adsorption. The amorphous silica prevents the montmorillonite from swelling and allows column packing. Protein adsorption was performed at different pH under static conditions. Furthermore, static capacities were determined. The material reveals high adsorption capacities for proteins under static conditions (270-408 mg/g), whereby proteins are mainly adsorbed via electrostatic interactions. The Freundlich isotherm is suggested as an adsorption model. For desorption a pH shift was found to be most effective. Binding and elution of human serum albumin and ovalbumin were tested under dynamic conditions. Dynamic capacities of about 40 mg/g for ovalbumin at 764 cm/h were found. The clay mineral provides suitable properties for the application as cost-efficient, alternative separation material.

Sensors in disposable bioreactors status and trends.

For better control of productivity and product quality, detailed monitoring of various parameters is required. Since disposable bioreactors become more and more important for biotechnological applications, adequate sensors for this type of reactor are necessary. The required properties of sensors used in disposable reactors differ from those of sensors for multiuse reactors. For example, sensors which are in direct contact with the medium must be inexpensive, but do not need a long life-time, since they can be used only once.This chapter gives an overview on the state of the art and future trends in the field of sensors suited for use in disposable bioreactors. The main focus here is on in situ sensors, which can be based on optical, semiconductor and ultrasonic technologies, but current concepts for disposable sampling units are also reviewed.