Using Fluorescence-Based Sensing to Accelerate Process Development - A prove-free system monitors accurately at very small scale - BioPharm International


Using Fluorescence-Based Sensing to Accelerate Process Development
A prove-free system monitors accurately at very small scale

BioPharm International
Volume 22, Issue 6


Despite the focus on the screening phases of the development cycle, this sensing technology also has value during the later phases of the development and production cycle. Recent trends tend toward the use of disposables during these phases because of lower operation and validation costs.1 Disposables primarily take the form of bag type bioreactors—plastic pillows that can be charged with a few to many hundreds of liters of cell culturing media. Because they are generally shipped sterile and disposed of after use, they require less labor during setup and teardown of the culturing system than conventional stirred-tank reactors. However, these bioreactors require the same careful monitoring as the conventional bioreactors. If conventional probe-type sensors are adapted for use in these disposable bioreactors, the system is then only "semi-disposable" because the sensors must be sterilized before and after use, and calibrated during use. To address this issue, some recent efforts have been made to develop miniature disposable probe-type sensors so that the entire system becomes truly disposable. These sensors, however, are still relatively costly for a disposable system, and fail to adequately address calibration issues and the consequent labor and time requirements.

A different approach to fully disposable systems that has been adopted by several manufacturers is the incorporation of fluorescence-based sensors in disposable-bag bioreactors.2 The sensor technology is applicable to any size bioreactor because it uses no volume-dependent components. The sensing patches themselves may be placed in the bags during fabrication and then sterilized by irradiation or by autoclaving, and then sealed into a presterilized bag. Alternately, the sensing patches can be autoclaved on a carrier (a clear glass or autoclaveable plastic slide or disk) that can then be inserted into a pocket in the bag before final sealing. This latter technique has the added advantage of facilitating alignment of the sensing patch in the bag with respect to the sensing head or fiber. The patches, which cost only a few dollars, are far less costly than even a disposable miniature probe and can be disposed of along with the bag after use. The sensing head itself remains ready to use again. In addition to the cost savings resulting from the disposal of only a very inexpensive patch, elimination of the effective need for calibration saves substantial time and labor costs associated with the culturing process.


Given the many advantages to fluorescence-based sensing, it is only natural to ask why this technology, which is nearly three-decades old has been so slow to catch on and why, even now, its use is primarily associated with small-scale apparatus.3

There are several factors that have contributed to its slow adoption. First, the biotech community is notoriously slow to embrace new technologies, particularly during the pilot and manufacturing phases of a drug-development cycle. This is because the cost of drug development and approval often greatly outweighs the cost of drug production, and the elapsed time required for drug approval is often far greater than the time required for screening and laboratory-scale operations. The use of a decades-old technology provides the manufacturer with a certain level of assurance during the approval process, and might therefore be preferred over a newer technology even if the newer technology is less costly and more efficient.

Second, the fluorescence-based instruments are just now reaching a level of robustness and affordability that makes them suitable for use in bioprocessing. Although conventional probes are subject to inaccuracy because of drift over time, this artifact can be virtually eliminated through frequent and careful calibration of the probe, a time-consuming operation but one that is effective nonetheless. In contrast, early fluorescence-based probes had several sources of error: inherent electronic variance from sensor to sensor, variability of patch formulation from batch-to-batch, and the need to ensure that the vessel being used conformed to certain specifications. In recent years, these problems have been substantially addressed: by including a microprocessor on board each instrument, the electronics can be normalized from sensor to sensor, largely mitigating electronic variance. Patch-to-patch variation is addressed by associating a calibration code with each batch of patches, similar to the codes used with most diabetic test strips, and the measurement results have been shown to be largely independent of bioreactor-to-bioreactor variance. The referenced testing was done using the Fluorometrix CellStation HTBR-1 12 station system.4

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