Scale-Up and Comparison Studies Evaluating Disposable Bioreactors and Probes - Process performance was comparable across all scales, and fiber optic sensors appeared interchangeable with conventional

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Scale-Up and Comparison Studies Evaluating Disposable Bioreactors and Probes
Process performance was comparable across all scales, and fiber optic sensors appeared interchangeable with conventional probes.


BioPharm International Supplements


Results

Process Optimization


Table 1. Data for a pH drift or no pH drift, and for a temperature shift mid-culture or no temperature shift, averaged over six runs of 2-L and 5-L glass vessel reactors for each condition.
The pH set point was optimized for the cell culture process. Set points in 0.2 increments were tested and the one that resulted in the highest peak viable cell density and final protein concentration was chosen. For product 1, the natural tendency of the cell line is an increase in pH toward the end of the culture. To maintain a lower set point, more CO2 must be added to the culture, which increases the pCO2 in the culture. High pCO2 levels have been shown to negatively affect the quality of product 1. To decrease the amount of pCO2 in the culture, experiments were run allowing the pH to drift after day nine. The pH drift was performed by removing the base control and increasing the set point, to ensure that the pH did not drift too high. Viable cell density, viability, and protein concentration were monitored throughout the culture and compared to runs in which the pH was maintained at the set point. Figure 1 shows the normalized pCO2 values over time in culture for 2- and 5-L reactors with and without a pH drift. Initially, the pH of the media is higher and must be brought down to the set point, accounting for higher pCO2 values in the vessels in the first few days. Around day nine, as the pCO2 increases again as a result of the natural tendency of the cells, the cultures with the drift show a decrease in pCO2. Cultures maintained at the set point show increased pCO2 over the same time. Table 1 shows average normalized peak viable cell density values, final viabilities, and normalized final protein concentrations for both conditions. Those values do not vary significantly, which indicates that letting the pH drift does not have a negative effect on the cell density or protein concentration, although it does decrease the pCO2 in the culture.


Figure 1. Normalized partial pressure of carbon dioxide (pCO2) versus time in culture for 2-L and 5-L reactors with and without a pH drift after day nine
Another parameter that was optimized was temperature. Changing the temperature set point to a lower value mid-culture has been shown to sustain higher cell viabilities.1 Higher cell viabilities make harvest easier because there is less cell debris. Experiments with a temperature shift mid-culture were performed and compared to runs with the temperature maintained at the set point. Figure 2 shows fractional cell viabilities over time in culture for runs with and without a temperature shift. The viabilities trend similarly through the exponential growth phase at the start of the culture. As the cell density achieves its maximum value, however, the viability begins to drop for the experiments with temperature maintained at the set point. For those that received a temperature shift, the viability is sustained at higher values through day 14, most likely because of slower cell growth. Table 1 displays average values for normalized peak viable cell density, final cell viability, and normalized final protein concentration for both sets of runs. The normalized peak viable cell density and normalized final protein concentration values did not vary significantly. However, the final viabilities were significantly different (p <0.01), showing that a temperature shift mid-culture is preferred.


Figure 2. Fractional viability versus time in culture for 2-L and 5-L reactors with and without a temperature shift mid-culture
In addition to process optimization, scale-up studies were performed to transfer from the 2- and 5-L scale up to the 1,000-L scale. The oxygen mass transfer coefficient (kLa) is an important value in scale-up studies and can vary depending on sparger type (i.e., pinhole or micro). By adjusting agitation speeds and air flow rates, kLa can be compared across scales. Studies were performed at the 2-L, 5-L, and 50-L scales. Optimal flow rates and agitation speeds were determined by achieving similar kLa values across scales.

Taking into account all parameters optimized in the 2- and 5-L vessels, the final process to transfer and scale-up to the SUBs was a 14 day fed-batch culture with a day 2 feed of 12% of the working volume, a temperature shift mid-culture, and a pH drift after day 9 when necessary. The SUBs have a dual sparger option (open pipe or frit). The open pipe sparger provides air to the culture while stripping excess CO2. If excess CO2 is stripped, the pH drift may not be necessary because the pCO2 levels will be decreased.


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