The plug-and-play online Raman spectrometer PAT tool empowers Culture Biosciences with real-time control.

Maverick: A multi-parameter PAT tool that’s plug-and-play and enables online monitoring in just a few minutes.

The plug-and-play online Raman spectrometer PAT tool empowers Culture Biosciences with real-time online control.

2023-12-12

Maverick: A multi-parameter PAT tool that’s plug-and-play and enables online monitoring in just a few minutes.

Based on Raman spectroscopy principles and three built-in models, this system can simultaneously and in real time measure three key process parameters—glucose, lactate, and total cell density (TCD)—across the entire validated range of CHO and HEK293 cell cultures. It can also connect up to six reactors simultaneously and display real-time data on a built-in screen or via a mobile app, helping to overcome the high upfront costs associated with conventional Raman-based methods. Compared to traditional Raman PAT instruments, the combination of an independent probe and spectrometer enables simultaneous, parallel scanning of multiple bioreactors. With OPC-UA feedback control and compliance with 3Q and 21 CFR Part 11 standards, this system facilitates rapid automation—from process development and process control through to pilot-scale production scaling.

BIOPROCESSING 4.0: Leveraging advanced digital technologies, automation, and data-driven approaches to transform traditional biopharmaceutical manufacturing processes. To enable innovative BIOPROCESSING 4.0 process development using PAT (Process Analytical Technology) methods, Culture Biosciences and 908 Devices have collaborated to test MAVERICK and Culture Biosciences’ cloud-connected 250-mL bioreactor. MAVERICK provides real-time, online analysis and control of bioprocesses—ready to use out of the box—with none of the costs, complexity, or risks associated with conventional Raman-based methods.

(Left image) A view of the Culture Biosciences Cloud bioreactor laboratory, equipped with two bioreactors (288 and 289) and a 908 Devices MAVERICK. (Right image) An enlarged view of the online probe inserted into a 250 mL reactor.

Cell Culture Protocol: Chinese Hamster Ovary (CHO) mammalian cells were cultured in four 250-mL bioreactors. Two of the bioreactors were equipped with MAVERICK (908 Devices) as experimental controls. Throughout the entire culture process, MAVERICK continuously and in real time measured glucose, lactate, and biomass levels. Under the control of Culture Biosciences’ cloud-connected proprietary controller and monitoring technology, the bioreactors were fully automated to receive feed media, glucose, and other nutrients. The feeding steps could be activated either manually or automatically, depending on the specific application requirements and customer workflows. In the tested protocol, feedback from daily offline measurements was used to adjust the glucose levels in the bioreactors via feed control. Sterile samples were taken from the bioreactors every 24 hours and analyzed for glucose, lactate, other metabolites (Flex2), and cell density (Vi-cell BLU). At the end of the experiment (14 days), data from both offline and online devices, as well as application programming interfaces (APIs), were analyzed using Culture’s console to evaluate the performance of the probes in Culture’s 250-mL bioreactor system.

Glucose: The results show that the offline data from Flex2 are consistent with the online PAT values from MAVERICK (RMSE = 0.32 g/L). The figure below demonstrates that MAVERICK, as an online PAT tool, accurately reflects the cell culture conditions within the bioreactor. Online PAT measurements enable real-time tracking of the effects of feed and glucose supplementation (blue line) in the bioreactor. These dynamic changes in cell culture conditions are not captured by the daily offline sample measurements (orange line).

MAVERICK online measurement of glucose (blue) in the Culture Biosciences bioreactor, and daily offline glucose measurements from Flex2 (orange).

The bioreactor from Culture Biosciences achieves accurate volume monitoring through cloud-connected sensors and controllers, as well as a data aggregation backend. Real-time monitoring of this data enables precise tracking of all sugar-feeding and nutrient-addition events throughout the entire experimental process. Based on the generated feed-volume dataset, the increase in glucose concentration observed during online MAVERICK measurements was explained.

The glucose peak measured by MAVERICK is consistent with the feed pump data captured and processed by the cloud bioreactor from Culture Biosciences.

Lactic acid: Similar to the observed glucose concentrations, MAVERICK can also accurately measure lactic acid levels compared to offline analysis (RMSE = 0.29 g/L).

Lactate measured online via the PAT tool (blue) and lactate measured via daily offline samples (orange).

Biomass: When these values are compared with the total cell density measured offline by the Vi-CELL BLU, Raman-based techniques typically require calibration curves for conversion into physical units. The figure below shows the raw signal measurements of MAVERICK (in biomass units, BMU) on the left y-axis and the total cell density measured by the Vi-CELL BLU (in millions of cells/mL) on the right y-axis. The figure demonstrates that the two curves are highly consistent, indicating that MAVERICK can effectively capture the underlying physical signals. The discrepancy between the two measurement results occurs after 250 hours, which can be attributed to the declining viability of mammalian cell cultures in the later stages of the experiment. A decrease in cell viability leads to an increase in cellular debris, which may confound the online PAT measurements. In contrast, the visual measurement provided by the Vi-CELL BLU can be adjusted to exclude cells undergoing lysis from the measurement, thus accounting for the observed difference between the two sets of results.

Comparison of biomass measured online by MAVERICK (arbitrary units; blue, left y-axis) with total cell density measured offline daily (millions of cells/mL; orange, right y-axis).

Conclusion: These experiments demonstrate that by using Culture Bioscience’s bioreactor in conjunction with MAVERICK, a novel Raman-spectroscopy–driven online PAT tool, it is possible to achieve real-time monitoring of multiple key analytical parameters.

A bioreactor combined with PAT tools can accelerate the understanding of process dynamics through real-time, continuous data streams. This continuous data flow also facilitates precise monitoring and control of cell culture processes, enabling the realization of BIOPROCESSING 4.0.

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