Automated and enhanced clone screening using a fully automated microtiter plate-based system for suspension cell culture.

Recently, we established an automated microtiter plate (MTP)-based system for suspension cell culture for high-throughput (HT) applications in biopharmaceutical process development. In the present report, the new system was evaluated regarding its potential to improve clone screening by allowing high-throughput fed-batch cultivation at an early stage. For this purpose, a fully automated procedure was compared to a mainly batch mode-based manual standard process. The new system performed daily measurements of viable cell density and product concentration for a total of 96 clones in biological duplicates that were evaluated for final clone selection. This resulted in a more than fivefold increase in sample throughput and 4 weeks of time saving compared to the reference process. The top clone characterized by the highest cell specific productivity was identified only by the new process. In contrast, this clone was lost in the expansion phase of the reference procedure. Overall, the new system identified more high-productive clones, offering more alternatives and flexibility for process development. In-process monitoring of glucose and lactate levels representing crucial secondary selection criteria further enhanced top clone identification. Clone characterization at an early stage was further extended by linking the MTP-based cell culture system to additional HT-analytic systems for N-glycosylation analysis as well as gene expression analysis by reverse transcriptase-quantitative polymerase chain reaction. These powerful tools connected to the automated MTP-based cell culture system lead to considerably advanced quality and speed of clone screening, and increase the probability of selecting the most suitable clone. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2760, 2019.

Establishment of a fully automated microtiter plate-based system for suspension cell culture and its application for enhanced process optimization.

We developed an automated microtiter plate (MTP)-based system for suspension cell culture to meet the increased demands for miniaturized high throughput applications in biopharmaceutical process development. The generic system is based on off-the-shelf commercial laboratory automation equipment and is able to utilize MTPs of different configurations (6-24 wells per plate) in orbital shaken mode. The shaking conditions were optimized by Computational Fluid Dynamics simulations. The fully automated system handles plate transport, seeding and feeding of cells, daily sampling, and preparation of analytical assays. The integration of all required analytical instrumentation into the system enables a hands-off operation which prevents bottlenecks in sample processing. The modular set-up makes the system flexible and adaptable for a continuous extension of analytical parameters and add-on components. The system proved suitable as screening tool for process development by verifying the comparability of results for the MTP-based system and bioreactors regarding profiles of viable cell density, lactate, and product concentration of CHO cell lines. These studies confirmed that 6 well MTPs as well as 24 deepwell MTPs were predictive for a scale up to a 1000 L stirred tank reactor (scale factor 1:200,000). Applying the established cell culture system for automated media blend screening in late stage development, a 22% increase in product yield was achieved in comparison to the reference process. The predicted product increase was subsequently confirmed in 2 L bioreactors. Thus, we demonstrated the feasibility of the automated MTP-based cell culture system for enhanced screening and optimization applications in process development and identified further application areas such as process robustness. The system offers a great potential to accelerate time-to-market for new biopharmaceuticals. Biotechnol. Bioeng. 2017;114: 113-121. © 2016 Wiley Periodicals, Inc.

A novel in situ probe for oxygen uptake rate measurement in mammalian cell cultures.

The newly developed in situ oxygen uptake rate (in situ OUR) probe presented in this article is based on the in situ microscope technology platform. It is designed to measure the oxygen uptake rate (OUR) of mammalian cells, an important parameter for metabolic flux analysis, inside a reactor (in situ) and in real-time. The system isolates a known volume of cell culture from the bulk inside the bioreactor, monitors the oxygen consumption over time, and releases the sample again. The sample is mixed during the measurement with a new agitation system to keep the cells in suspension and prevent oxygen concentration gradients. The OUR measurement system also doubles as a standard dissolved oxygen (DO) probe for process monitoring when it is not performing OUR measurements. It can be equipped with two different types of optical sensors (i.e., DO, pH) simultaneously or a conventional polarographic DO-probe (Clark type). This new probe was successfully tested in baby hamster kidney perfusion cell cultures.

Optical inline measurement procedures for counting and sizing cells in bioprocess technology.

To observe and control cultivation processes, optical sensors are used increasingly. Important parameters for controlling such processes are cell count, cell size distribution, and the morphology of cells. Among turbidity measurement methods, imaging procedures are applied for determining these process parameters. A disadvantage of most previously developed imaging procedures is that they are only available offline which requires sampling. On the other hand, available imaging inline probes can so far only deliver a limited number of process parameters. This chapter presents new optical procedures for the inline determination of cell count, cell size distribution, and other parameters. In particular, by in situ microscopy an imaging procedure will be described which allows the determination of direct and nondirect cell parameters in real time without sampling.

Logistic equations effectively model Mammalian cell batch and fed-batch kinetics by logically constraining the fit.

A four-parameter logistic equation was used to fit batch and fed-batch time profiles of viable cell density in order to estimate net growth rates from the inoculation through the cell death phase. Reduced three-parameter forms were used for nutrient uptake and metabolite/product formation rate calculations. These logistic equations constrained the fits to expected general concentration trends, either increasing followed by decreasing (four-parameter) or monotonic (three-parameter). The applicability of this approach was first verified for Chinese hamster ovary (CHO) cells cultivated in 15-L batch bioreactors. Cell density, metabolite, and nutrient concentrations were monitored over time and used to estimate the logistic parameters by nonlinear least squares. The logistic models fit the experimental data well, supporting the validity of this approach. Further evidence to this effect was obtained by applying the technique to three previously published batch studies for baby hamster kidney (BHK) and hybridoma cells in bioreactors ranging from 100 mL to 300 L. In 27 of the 30 batch data sets examined, the logistic models provided a statistically superior description of the experimental data than polynomial fitting. Two fed-batch experiments with hybridoma and CHO cells in benchtop bioreactors were also examined, and the logistic fits provided good representations of the experimental data in all 25 data sets. From a computational standpoint, this approach was simpler than classical approaches involving Monod-type kinetics. Since the logistic equations were analytically differentiable, specific rates could be readily estimated. Overall, the advantages of the logistic modeling approach should make it an attractive option for effectively estimating specific rates from batch and fed-batch cultures.

In-situ microscopy: Online process monitoring of mammalian cell cultures.

The in-situ microscope is a system developed to acquire images of mammalian cells directly inside a bioreactor (in-situ) duringa fermentation process. It requires only minimal operator intervention and it is well suited for either batch or long-termperfusion fermentation runs. The system fits into a 25 mm standard port and has a retractable housing, similar to the industry standard InTrac. Therefore, it can be cleaned and serviced without interruption of the process or risking contamination. A sampling zone inside the bioreactor encloses adefined volume of culture and an image sequence is taken. The height of the sampling zone is set by the control program and canbe adjusted during the cultivation to accommodate a wide range of change in cell density. The system has an infinity correctedoptical train and uses a progressive scan CCD camera to acquirehigh quality images. Process relevant information like cell density is extracted fromthe images by digital image processing software, currently in development for mammalian cells (CHO, BHK). The first version ofthe software will be able to estimate the cell density, cellsize distribution and to give information of the degree of aggregation (single and double cells, cell clusters).