Equal mixing time enables scale-down and optimization of a CHO cell culture process using a shaken microbioreactor system.

The advancement of microbioreactor technology in recent years has transformed early- and mid-stage process development. The monitoring and control capabilities of microbioreactors not only promote the quick accumulation of process knowledge but has also led to an increased scalability when compared to traditionally used systems such as shake flasks and microtitre plates. This study seeks to establish a framework for the micro-Matrix microbioreactor (Applikon-Biotechnology BV) as process development tool. Using the Dual Indicator System for Mixing Time, the system was initially characterized for mixing properties at varying operating conditions, which was found to yield mixing times between 0.9 and 41.8 s. A matched mixing time was proposed as scale-down criterion for an IgG4 producing GS-CHO fed-batch process between a 5 L stirred tank reactor (STR) and the micro-Matrix microbioreactor. Growth trends, maximum viable cell concentrations, final titre, and glycoprofiles were nearly identical at both scales. The scale-down model was then employed to optimize a bolus feeding regime using response surface methodology, which led to a 25.4% increase of the space-time yield and a 25% increase of the final titre. The optimized feeding strategy was validated at the small-scale and successfully scaled up to the 5 L STR. This work for the first time provides a framework of how the micro-Matrix microbioreactor can be implemented in a bioprocess development workflow and demonstrates scalability of growth and production kinetics as well as IgG4 glycosylation between the micro-Matrix and a benchtop-scale STR system.

Model-based workflow for scale-up of process strategies developed in miniaturized bioreactor systems.

Miniaturized bioreactor (MBR) systems are routinely used in the development of mammalian cell culture processes. However, scale-up of process strategies obtained in MBR- to larger scale is challenging due to mainly non-holistic scale-up approaches. In this study, a model-based workflow is introduced to quantify differences in the process dynamics between bioreactor scales and thus enable a more knowledge-driven scale-up. The workflow is applied to two case studies with antibody-producing Chinese hamster ovary cell lines. With the workflow, model parameter distributions are estimated first under consideration of experimental variability for different scales. Second, the obtained individual model parameter distributions are tested for statistical differences. In case of significant differences, model parametric distributions are transferred between the scales. In case study I, a fed-batch process in a microtiter plate (4 ml working volume) and lab-scale bioreactor (3750 ml working volume) was mathematically modeled and evaluated. No significant differences were identified for model parameter distributions reflecting process dynamics. Therefore, the microtiter plate can be applied as scale-down tool for the lab-scale bioreactor. In case study II, a fed-batch process in a 24-Deep-Well-Plate (2 ml working volume) and shake flask (40 ml working volume) with two feed media was investigated. Model parameter distributions showed significant differences. Thus, process strategies were mathematically transferred, and model predictions were simulated for a new shake flask culture setup and confirmed in validation experiments. Overall, the workflow enables a knowledge-driven evaluation of scale-up for a more efficient bioprocess design and optimization.

Bioprocess considerations for T-cell therapy: Investigating the impact of agitation, dissolved oxygen, and pH on T-cell expansion and differentiation.

Adoptive T-cell therapy (ACT) has emerged as a promising new way to treat systemic cancers such as acute lymphoblastic leukemia. However, the robustness and reproducibility of the manufacturing process remains a challenge. Here, a single-use 24-well microbioreactor (micro-Matrix) was assessed for its use as a high-throughput screening tool to investigate the effect and the interaction of different shaking speeds, dissolved oxygen (DO), and pH levels on the growth and differentiation of primary T cells in a perfusion-mimic process. The full factorial design allowed for the generation of predictive models, which were used to find optimal culture conditions. Agitation was shown to play a fundamental role in the proliferation of T cells. A shaking speed of 200 rpm drastically improved the final viable cell concentration (VCC), while the viability was maintained above 90% throughout the cultivation. VCCs reached a maximum of 9.22 × 106 cells/ml. The distribution of CD8+ central memory T cells (TCM ), was found to be largely unaffected by the shaking speed. A clear interaction between pH and DO (p < .001) was established for the cell growth and the optimal culture conditions were identified for a combination of 200 rpm, 25% DO, and pH of 7.4. The combination of microbioreactor technology and Design of Experiment methodology provides a powerful tool to rapidly gain an understanding of the design space of the T-cell manufacturing process.

Using a Parallel Micro-Cultivation System (Micro-Matrix) as a Process Development Tool for Cell Culture Applications.

Micro-bioreactors appear frequently in today's biotechnology industry as screening and process development tools for cell culture applications. The micro-bioreactor's small volume allows for a high throughput, and when compared to other small-scale systems, such as microtiter plates, its measurement and control capabilities offer a much better insight into the bioprocess. Applikon's micro-Matrix is one of the micro-bioreactors that are commercially available today. The micro-Matrix system consists of shaken disposable 24 deep square well plates in which each well is controlled individually for pH, dissolved oxygen (DO), and temperature. Additionally, a feeding module supports automated additions of liquid to each well. This chapter describes how the micro-Matrix can be used for fed-batch cultivations of Chinese Hamster Ovary (CHO) cells.

Towards the development of automated fed-batch cell culture processes at microscale.

Aim: To investigate the impact of various feeding strategies on the growth and productivity of a GS-CHO cell line. Methods: Feed additions were conducted at fixed volumes or linked to a marker such as cell growth or metabolism and added as bolus or near-continuously using the automated feeding module of the micro-Matrix (Applikon). Results: The selected feeding regimens supported maximum viable cell densities of up to 1.9 × 107 cells ml-1 and final titers of up to 1.13 g l-1. Differences in growth and titer between feeding strategies were insignificant, with the exception of one feeding strategy. Conclusion: As the more complex feeding strategies did not create an advantage, the selection of a simple feeding strategy such as bolus or continuous addition of feed medium is preferred.

A simple method to determine evaporation and compensate for liquid losses in small-scale cell culture systems.

OBJECTIVES: Establish a method to indirectly measure evaporation in microwell-based cell culture systems and show that the proposed method allows compensating for liquid losses in fed-batch processes. RESULTS: A correlation between evaporation and the concentration of Na+ was found (R2 = 0.95) when using the 24-well-based miniature bioreactor system (micro-Matrix) for a batch culture with GS-CHO. Based on these results, a method was developed to counteract evaporation with periodic water additions based on measurements of the Na+ concentration. Implementation of this method resulted in a reduction of the relative liquid loss after 15 days of a fed-batch cultivation from 36.7 ± 6.7% without volume corrections to 6.9 ± 6.5% with volume corrections. CONCLUSION: A procedure was established to indirectly measure evaporation through a correlation with the level of Na+ ions in solution and deriving a simple formula to account for liquid losses.