Oscar Platas Barradas

Evaluation of criteria for bioreactor comparison and operation standardization for mammalian cell culture

Development of bioprocesses with mammalian cell culture deals with different bioreactor types and scales. The bioreactors might be intended for generation of cell inoculum and production, research, process development, validation, or transfer purposes. During these activities, not only the difficulty of up and downscaling might lead to failure of consistency in cell growth, but also the use of different bioreactor geometries and operation conditions. In such cases, criteria for bioreactor design and process transfer should be carefully evaluated in order to select appropriate cultivation parameters.

Synchronized mammalian cell culture: part II--population ensemble modeling and analysis for development of reproducible processes.

The consideration of inherent population inhomogeneities of mammalian cell cultures becomes increasingly important for systems biology study and for developing more stable and efficient processes. However, variations of cellular properties belonging to different sub‐populations and their potential effects on cellular physiology and kinetics of culture productivity under bioproduction conditions have not yet been much in the focus of research. Culture heterogeneity is strongly determined by the advance of the cell cycle. The assignment of cell‐cycle specific cellular variations to large‐scale process conditions can be optimally determined based on the combination of (partially) synchronized cultivation under otherwise physiological conditions and subsequent population‐resolved model adaptation. The first step has been achieved using the physical selection method of countercurrent flow centrifugal elutriation, recently established in our group for different mammalian cell lines which is presented in Part I of this paper series. In this second part, we demonstrate the successful adaptation and application of a cell‐cycle dependent population balance ensemble model to describe and understand synchronized bioreactor cultivations performed with two model mammalian cell lines, AGE1.HNAAT and CHO‐K1. Numerical adaptation of the model to experimental data allows for detection of phase‐specific parameters and for determination of significant variations between different phases and different cell lines. It shows that special care must be taken with regard to the sampling frequency in such oscillation cultures to minimize phase shift (jitter) artifacts. Based on predictions of long‐term oscillation behavior of a culture depending on its start conditions, optimal elutriation setup trade‐offs between high cell yields and high synchronization efficiency are proposed.

Mammalian cell culture synchronization under physiological conditions and population dynamic simulation

The overall behavior of cell cultures is determined by the actions and regulations of all cells and their interaction in a mixed population. However, the dynamics caused by diversity and heterogeneity within cultures is often neglected in the study of cell culture processes. Usually, a bulk behavior is assumed, although heterogeneity prevails in most cases. It is, however, not valid to conclude from the bulk behavior to the single cell behavior. Instead, it is necessary to include the behavior and kinetics of subpopulations and their interactions into models in order to elucidate the dynamic effects occurring in typical cell cultures. Heterogeneity in cell cultures is largely caused by the progress of the cell cycle. Cell cycle-dependent dynamics resulting for example in variable transfection efficiencies or expression bistability have recently attracted attention. In order to elucidate cell cycle-dependent regulations in cell cultures, it is desirable to synchronize a culture with minimal perturbation, which is possible with different yield and quality using physical methods, but not possible for frequently used chemical, or whole-culture methods. Then, the culture is cultivated again under physiological conditions and subpopulation-resolved analysis and modeling approaches are applied. This should allow to account for the variable contributions of subpopulations to the whole behavior and also for obtaining hereto unaccessible dynamic information of cellular regulation. In this short review, we summarize techniques and key issues to be considered for successful synchronization, cultivation, and modeling in order to achieve the goal of better understanding cell culture at a population level.

Synchronized mammalian cell culture: part I--a physical strategy for synchronized cultivation under physiological conditions.

Conventional analysis and optimization procedures of mammalian cell culture processes mostly treat the culture as a homogeneous population. Hence, the focus is on cell physiology and metabolism, cell line development, and process control strategy. Impact on cultivations caused by potential variations in cellular properties between different subpopulations, however, has not yet been evaluated systematically. One main cause for the formation of such subpopulations is the progress of all cells through the cell cycle. The interaction of potential cell cycle specific variations in the cell behavior with large‐scale process conditions can be optimally determined by means of (partially) synchronized cultivations, with subsequent population resolved model analysis. Therefore, it is desirable to synchronize a culture with minimal perturbation, which is possible with different yield and quality using physical selection methods, but not with frequently used chemical or whole‐culture methods. Conventional nonsynchronizing methods with subsequent cell‐specific, for example, flow cytometric analysis, can only resolve cell‐limited effects of the cell cycle. In this work, we demonstrate countercurrent‐flow centrifugal elutriation as a useful physical method to enrich mammalian cell populations within different phases of a cell cycle, which can be further cultivated for synchronized growth in bioreactors under physiological conditions. The presented combined approach contrasts with other physical selection methods especially with respect to the achievable yield, which makes it suitable for bioreactor scale cultivations. As shown with two industrial cell lines (CHO‐K1 and human AGE1.HN), synchronous inocula can be obtained with overall synchrony degrees of up to 82% in the G1 phase, 53% in the S phase and 60% in the G2/M phase, with enrichment factors ( Ysync ) of 1.71, 1.79, and 4.24 respectively. Cells are able to grow with synchrony in bioreactors over several cell cycles. This strategy, combined with population‐resolved model analysis and parameter extraction as described in the accompanying paper, offers new possibilities for studies of cell lines and processes at levels of cell cycle and population under physiological conditions.

A New Integrated Lab-on-a-Chip System for Fast Dynamic Study of Mammalian Cells under Physiological Conditions in Bioreactor

For the quantitative analysis of cellular metabolism and its dynamics it is essential to achieve rapid sampling, fast quenching of metabolism and the removal of extracellular metabolites. Common manual sample preparation methods and protocols for cells are time-consuming and often lead to the loss of physiological conditions. In this work, we present a microchip-bioreactor setup which provides an integrated and rapid sample preparation of mammalian cells. The lab-on-a-chip system consists of five connected units that allow sample treatment, mixing and incubation of the cells, followed by cell separation and simultaneous exchange of media within seconds. This microsystem is directly integrated into a bioreactor for mammalian cell cultivation. By applying overpressure (2 bar) onto the bioreactor, this setup allows pulsation free, defined, fast, and continuous sampling. Experiments evince that Chinese Hamster Ovary cells (CHO-K1) can be separated from the culture broth and transferred into a new medium efficiently. Furthermore, this setup permits the treatment of cells for a defined time (9 s or 18 s) which can be utilized for pulse experiments, quenching of cell metabolism, and/or another defined chemical treatment. Proof of concept experiments were performed using glutamine containing medium for pulse experiments. Continuous sampling of cells showed a high reproducibility over a period of 18 h.

Physical methods for synchronization of a human production cell line

Background The study of central metabolism and the interaction of its dynamics during growth, product formation and cell division are key tasks to decode the complex metabolic network of mammalian cells. For this purpose, not only the quantitative determination of key cellular molecules is necessary, but also the variation of their expression rates in time, e.g. cell cycle dependent gene expression. Thus, synchronization of cultured cells is a requisite for almost any attempt to elucidate these time dependent cellular processes. Synchronous cell growth can help to gain deeper insight into dynamics of cellular metabolism. In our work, physical methods for synchronization of the human production cell line AGE1.HN (ProBioGen AG) are experimentally tested. Cell-size distribution, DNA-content and the number of synchronous divisions are used for comparison of the methods. According to our results, the enrichment of an AGE1. HN cell population within a cell cycle phase is possible. Currently, the increase of cell yield and the improvement of conditions for cell-growth resumption after synchronization are being studied.

Batch-to-batch variability of two human designer cell lines - AGE1.HN and AGE1.HN.AAT - carried out by different laboratories under defined culture conditions using a mathematical model

Systems biology approaches involve collaboration of a larger number of research groups. Experiments are being performed in different laboratories dealing with different aspects of the topic of interest. Therefore, comparability of data collected for further analysis and modeling needs critical assessment. Here, growth and product formation of two human designer cell lines (AGE1.HN and its α1‐antitrypsin producing clone AGE1.HN.AAT) were investigated by four research laboratories. Cell lines were cultivated in shake flasks and stirred tank bioreactors operated at standardized conditions using a chemically defined medium, and a simple mathematical model was used to estimate characteristic process parameters. Results obtained for 35 batches showed that neither the initial viable cell concentration nor the initial concentration of glucose and glutamine showed significant differences between laboratories. For these measurements with low variations, specific growth rate and yields varied between 8.5 and 26% (relative standard error), indicating comparability of cultivations between laboratories for exponential growth. Higher variations of fitted parameters related to measurements with high initial variation. Comparing the nonproducing with the producing cell line, no significant differences were found regarding growth dynamics and metabolism. Overall, it seems justified to draw conclusions based on the entire experimental dataset of this systems biology project.

Criteria for bioreactor comparison and operation standardisation during process development for mammalian cell culture

BackgroundDevelopment of bioprocesses for animal cells has to dealwith different bioreactor types and scales. Bioreactorsmight be intended for generation of cell inoculum andproduction, research, process development, validation ortransfer purposes. During these activities, not only thedifficulty of up- and downscaling might lead to failureof consistency in cell growth, but also the use of differ-ent bioreactor geometries and operation conditions. Insuch cases, the criteria for bioreactor design and processtransfer should be carefully evaluated in order to avoidan erroneous transfer of cultivation parameters.In this work, power input, mixing time, impeller tipspeed, and Reynolds number have been compared sys-tematically for the cultivation of the human cell lineAGE1.HN

DoE of fed-batch processes - model-based design and experimental evaluation.

Background Experimental process-development and optimization is expensive and time-consuming. Real optimization by means of design of experiments involves data generation before optimization can be aimed for. This can make the way from process development to process establishment even harder, since academia or start-up research facilities might not have the possibility to generate these data. Furthermore, bioprocesses involving mammalian cells deal with many critical variables; processes are not only carried out batch wise, but increasingly in fedbatch mode with desired feeding profiles. The use of DoE tools in combination with an appropriate growth model might allow the experimenter to develop and to test fed-batch strategies in silico, before experiments are carried out in the laboratory. In our work, an unstructured model for mammalian cell culture was used for simulation. Kinetic parameters were derived from a small number of shake-flask experiments. The model was tested for data generation on common fed-batch strategies. By means of design of experiments strategies, relevant conditions were selected and experimentally tested. In this way, suitable fed-batch strategies for mammalian cell lines are evaluated in silico before bioreactor experiments are to be performed. This results in a significant reduction in the number of experiments during process development for mammalian cell culture.

Characterisation of cultivation of the human cell line AGE1.HN.AAT

Background Human cell lines are an interesting alternative to CHO cells for the production of recombinant proteins and monoclonal antibodies, because of their ability to produce genuine human posttranslational modifications. The human cell line AGE1.HN.AAT (ProBioGen, Berlin, Germany), that originated from human neural precursor tissue, has been adapted to serum-free conditions and cultivated in many different systems. Here we present our results using this cell line in a scale-up of batch cultivation from 50 mL vented polypropylene tube on a shaking platform, polycarbonate shakeflask (cultivation volume from 50 mL up to 300 mL), a 2 L-glass vessel stirred tank reactor and a 20 L-stainless steel stirred tank reactor (both Sartorius Stedim, Goettingen, Germany).