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30 | SEPTEMBER 1, 2016 | GENengnews.com | Genetic Engineering & Biotechnology News Martin P. Debreczeny, Ph.D. The recent trend in bioprocess development toward small bioreactors and high cell den- sities presents challenges for accurate on- line monitoring of liquid cell cultures. Traditional on-line optical probes, such as transmission probes (also known as absor- bance or optical density probes) and reflec- tance probes (also known as back-scatter or turbidity probes), typically require a 12 mm or larger diameter port into the bioreactor vessel and are limited in their linear response to about one order of magnitude of cell bio- mass range. In small bioreactors, such as 250 mL capacity vessels, the number of available large diameter ports is necessarily limited. Monitoring cell biomass from inoculation (e.g. <0.1 g/L dry cell weight) to harvest (e.g. >100 g/L) frequently requires at least three orders of magnitude of cell biomass sensitivity. Several factors contribute to the lim- ited range of linear response of traditional transmission and reflectance probes. For reflectance probes, a frequent complication at low cell density is interference from re- flective objects, such as impellers or other probes in the bioreactor. Such interference can lead to an S-shaped response curve, wherein interference at low biomass is grad- ually overtaken by reflectance from cells at higher biomass. For absorbance probes, the well-known break-down of the Beer-Lambert law is manifested as a flattening of the absor- bance curve at high concentrations. A past approach to extending the useful range of optical probes has been to fit a curve to the response beyond the linear range. But this technique suffers from increasing inaccuracy as the response increasingly deviates from linearity, especially if the causes of nonlinear- ity are not consistent across bioreactor runs. Extending the Linear Response Range The center of the linear range of an opti- cal probe is determined by the path length that light must travel through the medium to get from the source to the detector. Decreas- ing this source-detector separation has the effect of shifting the linear response range to higher biomass. One approach to extending the range of biomass is to use several probes with different path lengths to monitor the same process. This technique is typically precluded in small-scale bioreactors due to space limitations. An alternative approach is to measure cell biomass reflectance through the bioreactor wall, such as the BE2100 sensor manufac- tured by BugLab. This noninvasive method eliminates the risk of contamination from the Optimizing Sensor Linear Response Range Under Challenging Process Conditions On-Line Optical Cell Biomass Monitoring BIOPROCESSING Tech Note This graph shows the optical reflectance of a liquid culture of Saccharomyces cerevisiae as a function of yeast dry cell weight. The culture was maintained in a 250 mL bioreactor, and optical reflectance was measured at 1310 nm. Notice that both optical reflectance and yeast dry cell weight vary over four orders of magnitude. mee What mou mave Been missing mimple Western mesƟng mervices GLP & ISO CerƟfied Ti Tired of running Western blots? Leave validaƟng anƟbodies and opƟmizing condiƟons to us. RayBiotech's Simple Western Service uses state-of-the-art capillary immunobloƫng to achieve ng to pg sensiƟvity with as liƩle as 10 µg of starƟng material. TesƟng can be done with with an anƟbody from RayBiotech's vast pre-validated anƟbody library or a third party anƟbody of your choice. p-mBmm mesƟng Data Lane 1: Standard ladder Lane 2: T47D, cell lysate Lane 3: T47D + UV treatment, cell lysate Primary: Mouse anƟ-p-NBS1 Se Secondary: HRP-conjugated goat anƟ-mouse IgG raybiotech.com mmmmmmm mmm mmmmmmmmmm mmmmmmmmm mmmmm mmmm +1.888.494.8555