Application Note - Single-Use Viable Cell Density Measurement in Rocking Motion Bioreactors

The ZHAW is one of the leading Swiss universities of applied sciences, with main expertise in life sciences and facility management, particularly in the areas of environment, food, and health. They make vital contributions to solving social challenges and improving quality of life. Their success is based on five dynamic institutes with extensive competence in research, development, and services in the disciplines of chemistry and biotechnology, food and beverage innovation, natural resource sciences, applied simulation, and facility management.

The ZHAW collaborated with Hamilton to conduct extensive trials and develop the feasibility of their viable cell density (VCD) measurement technology in rocking motion bioreactors. While Hamilton’s VCD measurement technology has been established in stirred tank bioreactors, it faces challenges in rocking motion bioreactor systems where the sensing element may only be partially covered by the medium, leading to noise in the measurement signal and affecting stability and accuracy. Through their collaboration, the ZHAW worked closely with Hamilton to address these challenges and optimize the VCD measurement technology for use in rocking motion bioreactors, contributing to the advancement of bioprocessing technologies in the industry.

The bioprocessing industry is trending towards integrated continuous manufacturing, which demands robust process analytical methods (PAT) such as viable cell density (VCD) measurement.  VCD is a critical performance indicator as it directly correlates with the final product yield by indicating the amount of active and producing cells. While other important parameters in bioprocessing, such as product titer, cell viability, product quality attributes, and process efficiency, also contribute to process performance, this application note focuses on the significance of VCD. The reliable measurement of viable cell density (VCD) is influenced by several factors, one of which is the sufficient coverage of the sensing element with media to measure. The coverage is influenced by different factors like bag size, filling volume of the bag, media properties (e.g., conductivity) and also the rocking motion speed which again is influenced by the rocking angle and the rocks per minute of the rocker. Also, the position of the patch in the bag is influencing the measurement. This is optimized from the bag manufacturer.

Hamilton’s new VCD sensor is designed to detect favorable measuring conditions and exclude impedance errors while providing a warning when process conditions do not allow sufficient measurement accuracy. In a collaboration with ZHAW, the signal stability of the SU VCD measurement system was investigated using Cytiva’s ReadyToProcess Wave  25 Rocker platform and non-sterile custom bags that mimic cultivation conditions with sodium chloride solution. With this conductive solution but zero permittivity, the working range of the rocker can be elaborated as the conductivity measurement indicates the measurement error best. The investigation demonstrated excellent measurement performance, even at low filling volumes of 20% of the bag volume, using the Hamilton measurement system, which was also comparable to a competitor system in terms of volume and rocking motion speed. The powerful electronics of the Hamilton VCD system allow for short measurement cycles and a wider process window.

To confirm the theoretical determination of the working range, an IgG-producing ExpiCHO-S cell line (Gibco) was cultivated using the Hamilton VCD system on Cytiva’s ReadyToProcess Wave 25 Rocker platform and sterile custom bags (see figure 3) with an initial working volume of 2 L and a starting cell density of 0.3x10e6 cells/mL. Two feedings of 1.5 L each were conducted during the 8-day run, and the online VCD readings were compared to offline readings using a cell culture analyzer (figure 4). The process was terminated after the VCD dropped below 0.5x10e6 cells/mL which allowed also the collection of data during the stationary and death phase of the cells. The cultivation process was repeated to show the precision and compared to two processes with the competitor system, achieving peak cell densities of 17.5x10e6 cells/mL and 18.9x10e6 cells/mL, respectively (see figure 5). The peak on the online permittivity during the apoptotic phase (death phase) is appearing due to the misinterpretation of half-dead cells as living cells. This peak can be used as a indication for the harvesting time. The results demonstrate that the Hamilton SU VCD measurement system works optimally even at low filling volumes and achieves comparable results to the competition system. The results provided in the cooperation with ZHAW demonstrate the optimal working conditions for the Hamilton SU VCD measurment system as low as down to 20% of the bag volume. During the two cultivations, the online VCD signal corresponded well with the offline measured values.

The Incyte P SU sensor is not only robust and durable but also features an intuitive design for easy plug and play handling. When combined with the user-friendly ArcAir software, the Incyte P SU sensor allows for real-time monitoring of the viable cell density during cultivation. This capability can lead to improved controllability and performance of bioprocesses, making the Incyte P SU sensor an essential tool for bioprocessing applications.

Authors: Géraldine Gubser, Yannick Senn, Iris Poggendorf, Zurich University of Applied Sciences