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A bench scale cell culture model representative of manufacturing scale (2,000 L) was developed based on oxygen mass transfer principles, for a CHO-based process producing a recombinant human protein. Cell culture performance differences across scales are characterized most often by sub-optimal performance in manufacturing scale bioreactors. By contrast in this study, reduced growth rates were observed at bench scale during the initial model development. Bioreactor models based on power per unit volume (P/V), volumetric mass transfer coefficient (kL a), and oxygen transfer rate (OTR) were evaluated to address this scale performance difference. Lower viable cell densities observed for the P/V model were attributed to higher sparge rates and reduced oxygen mass transfer efficiency (kL a) of the small scale hole spargers. Increasing the sparger kL a by decreasing the pore size resulted in a further decrease in growth at bench scale. Due to sensitivity of the cell line to gas sparge rate and bubble size that was revealed by the P/V and kL a models, an OTR model based on oxygen enrichment and increased P/V was selected that generated endpoint sparge rates representative of 2,000 L scale. This final bench scale model generated similar growth rates as manufacturing. In order to take into account other routinely monitored process parameters besides growth, a multivariate statistical approach was applied to demonstrate validity of the small scale model. After the model was selected based on univariate and multivariate analysis, product quality was generated and verified to fall within the 95% confidence limit of the multivariate model.

Escherichia coli, genetically engineered with a mercury(II)‐sensitive promoter and the lux genes from Vibrio fischeri, were used as microbial bioluminescent sensors for the detection of mercury. Evaluation of this genetic construction was carried out by determining the effects of various parameters on cell suspensions maintained at constant conditions in a small 100‐mL vessel. The strongest light intensities and quickest induction times occurred with cells in the midexponential growth phase maintained at 28°C, concentrated to 1 × 109 cells/mL, mixed at very fast speeds, and aerated at 2 vvm (volume of air per volume of culture per minute) during light measurement in the small vessel. The cells were sensitive to the mercuric ion in the range of 20 nM to 4 μM (4 to 800 ppb), and the total response time was on the order of 1 hour, depending on the above parameters. The cells exhibited great specificity for mercury. The cells had almost equal specificity for organic and inorganic forms of the mercuric ion and responded more weakly to the mercurous ion. A simple, inexpensive, durable miniature probe (3 mL) was constructed and operated using the optimum parameters found in the small vessel as a guide. The range of sensitivity to the mercuric ion detected in the probe was 10 nM to 4 μM when aeration was provided. © 1993 John Wiley & Sons, Inc.

There are three main potential sources for cell shear damage existing in stirred tank bioreactors. One is the potential high energy dissipation in the immediate impeller zones; another from small gas bubble burst; and third is from high gas entrance velocity (GEV) emitting from the sparger. While the first two have been thoroughly addressed for the scale‐up of Chinese hamster ovary (CHO) cell culture knowing that a wide tolerable agitation range with non‐damaging energy dissipation exists and the use of shear protectants like Pluronic F68 guard against cell damage caused by bubble burst, GEV remains a potential scale‐up problem across scales for the drilled hole or open pipe sparger designs. GEV as high as 170 m/s due to high gas flow rates and relatively small sparger hole diameters was observed to be significantly detrimental to cell culture performance in a 12,000 L bioreactor when compared to a satellite 2 L bioreactor run with GEV of <1 m/s. Small scale study of GEV as high as 265 m/s confirmed this. Based on the results of this study, a critical GEV of >60 m/s for CHO cells is proposed, whereas previously 30 m/s has been reported for NS0 cells by Zhu, Cuenca, Zhou, and Varma (2008. Biotechnol. Bioeng., 101, 751–760). Implementation of new large scale spargers with larger diameter and more holes lowered GEV and helped improve the cell culture performance, closing the scale‐up gap. Design of such new spargers was even more critical when hole plugging was discovered during large scale cultivation hence exacerbating the GEV impact. Furthermore, development of a scale down model based on mimicry of the large scale GEV profile as a function of time was proven to be beneficial for reproducing large scale results.

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