Contribution to the development of methods and systems for the automatization during the early stages of bioprocess development

This thesis is framed within the field of red biotechnology and more specifically in the development of bioprocesses for cell species that feature some therapeutical interest, either for the production of vaccines and monoclonal antibodies or stem cell experimental research. The main objective was the development and application of different instrumental techniques for the control and online monitorization of cell cultures. Oxygen consumption OUR (Oxygen Uptake Rate) was chosen as the central theme since this parameter has often been referenced as the most straighforward indicator of metabolic activity in animal cell culture. This thesis was carried out in the context of a Spin-Off project (Hexascreen Culture Technologies) whose objective was the development of disposable Minibioreactors intended for biopharmaceutical research. Obviously, this has led to a number of important trade-offs, as well as the proposal of several imaginative solutions to solve various technological challenges. For this reason and in order to offer a better idea of the work's scope, it was decided to include in the thesis not only the description of the method and results related to the OUR estimation but a detailed description of the systems developed. Results demonstrate the feasibility of a simplified procedure for estimating the oxygen consumption. This is a review of the Stationary liquid phase mass balance method which allows reducing the implementation cost and unlike the Dynamic method (The most usual thechnique) prevents changes on the oxygen tension that could affect the cell's normal arctivity. The proposed method is based on the accurate control of the oxygen concentration by means of PWM driven electrovalves and using the control loop internal signals to estimate the OUR.Aquesta Tesi doctoral està enquadrada en l'àmbit de la Biotecnologia vermella i més concretament en el desenvolupament de Bioprocessos relacionats amb espècies cel·lulars d’interès terapèutic, bé sigui per a la producció de vacunes, anticossos monoclonals o bé per a la recerca experimental amb cèl·lules mare. L'objectiu general ha estat el desenvolupament i aplicació de diferents tècniques instrumentals per al control i monitorització en línia de cultius cel·lulars, tant mateix d'entre les diferents tècniques emprades es va escollir la monitorització de la demanda d'oxigen O.U.R. (Oxygen Uptake Rate) com a tema central de la tesi degut a que aquest paràmetre ha estat referenciat sovint com un dels millors indicadors de l'activitat metabòlica en cultius de cèl·lules animals. Cal mencionar que la Tesi ha estat duta a terme en el context d'un projecte empresarial (HexaScreen Culture Technologies) l'objectiu del qual ha estat el desenvolupament de Minibioreactors d'un sol ús orientats al mon de la recerca Biofarmacèutica. Òbviament això ha comportant un número important de compromisos a l'hora d'abordar les diferents tasques, així com el plantejament de solucions imaginatives per a la resolució dels diferents reptes tecnològic. Per aquest motiu i per tal de transmetre una millor idea de l'abast del treball realitzat, es va decidir incloure en la tesi no només la descripció del mètode i resultats relacionats amb l'estimació de la O.U.R. sinó amés una descripció prou detallada dels sistemes desenvolupats. Pel que fa al tema central de la tesi, es demostra la viabilitat d'un procediment simplificat per a l'estimació de la demanda d’oxigen. Es tracta d'una revisió del procediment d'estimació de la OUR en condicions de concentració estacionària en la fase líquida que permet reduir-ne el cost de implementació tot prescindint de l'ús de cabalímetres màssics, així com a diferència del mètode dinàmic (Tècnica més habitual) evitar cap mena de canvi en la tensió d’oxigen que pogués afectar l’activitat normal de les cèl·lules. El mètode proposat, es basa en el control de la concentració d’oxigen mitjançant actuació PWM de les vàlvules d'aereació i l’ús dels propis senyals del llaç de control per tal d'estimar la O.U.R.Postprint (published version

A simplified implementation of the stationary liquid mass balance method for on-line OUR monitoring in animal cell cultures

This is the peer reviewed version of the following article: [Fontova, A. , Lecina, M. , López‐Repullo, J. , Martínez‐Monge, I. , Comas, P. , Bragós, R. and Cairó, J. J. (2018), A simplified implementation of the stationary liquid mass balance method for on‐line OUR monitoring in animal cell cultures. J. Chem. Technol. Biotechnol. doi:10.1002/jctb.5551], which has been published in final form at [doi:10.1002/jctb.5551]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.BACKGROUND: Compared with other methods, the stationary liquid mass balance method for oxygen uptake rate (OUR) determination offers advantages in terms of estimation accuracy and reduction of stress. However, the need for sophisticated instrumentation, like mass flow controllers and gas analysers, has historically limited wider implementation of such a method. In this paper, a new simplified method based on inexpensive valves for the continuous estimation of OUR in animal cell cultures is evaluated. The determination of OUR values is based on accurate operation of the dissolved oxygen (DO) control loop and monitoring of its internal variables. RESULTS: The method developed was tested empirically in 2¿L bioreactor HEK293 batch cultures. OUR profiles obtained by a dynamic method, global mass balance method and the developed simplified method were monitored and compared. The results show how OUR profile obtained with the proposed method better follows the off-line cell density determination. The OUR estimation frequency was also increased, improving the method capabilities and applications. The theoretical rationale of the method was extended to the sensitivity analysis which was analytically and numerically approached. CONCLUSIONS: The results showed the proposed method to be not only cheap, but also a reliable alternative to monitor the metabolic activity in bioreactors in many biotechnological processes, being a useful tool for high cell density culture strategies implementation based on OUR monitoring.Peer ReviewedPostprint (published version

Salisbury College Art and design

SIGLEAvailable from British Library Document Supply Centre-DSC:7168.13917(1/2001) / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Electrical Impedance Spectroscopy cell monitoring in a miniaturized bioreactor

Peer Reviewe

Route data representation for ATC simulation experiments

SIGLEAvailable from British Library Document Supply Centre- DSC:5644.91(RSRE-M--4133) / BLDSC - British Library Document Supply CentreGBUnited Kingdo

A simplified implementation of the O.U.R. stationary liquid mass balance estimation method for on-line monitoring in animal cell production processes

Peer Reviewe

Real-time and on-line monitoring of morphological cell parameters using electrical impedance spectroscopy measurements

BACKGROUND: Acquisition of electrical impedance spectroscopy (EIS) measurements enables one to obtain information about the features of cell cultures, which can be applied for real-time and on-linemonitoring purposes. RESULTS: Impedance measurements were carried out in three different cell specimens with different sizes and shapes (Vero cells, hybridoma and Escherichia coli) at different stages of cell culture, as well as during a controlled detachment process of an adherent animal cell line. The relaxation spectra obtained were fitted to a Cole impedance model and the corresponding parameters analyzed. The use of a four-electrodes measurement system decreased the dependency on the electrode interface’s impedance, and thus resulted in a systemmore sensitive to the cell features. The EISmonitoring of different cultures expansion showed the expected inverse proportional relationship between the central relaxation frequency and the cell cross-sectional area. The morphological changes of fibroblast cells during the detachment processes were also studied. Interestingly, EIS displayed the proportional relationship between the ¿ parameter of the Cole impedance model and the cell shape dispersion from sphericalmorphology (considering spherical shape ideality). CONCLUSION: The results obtained reveal the potential for developing a real-time monitoring tool for cell morphology features such as cell size and shape, which are involved inmany cellular processes like cell expansion, differentiation, cell attachment or cell death.Peer ReviewedPostprint (author's final draft

Real-time and on-line monitoring of morphological cell parameters using electrical impedance spectroscopy measurements

BACKGROUND: Acquisition of electrical impedance spectroscopy (EIS) measurements enables one to obtain information about the features of cell cultures, which can be applied for real-time and on-linemonitoring purposes. RESULTS: Impedance measurements were carried out in three different cell specimens with different sizes and shapes (Vero cells, hybridoma and Escherichia coli) at different stages of cell culture, as well as during a controlled detachment process of an adherent animal cell line. The relaxation spectra obtained were fitted to a Cole impedance model and the corresponding parameters analyzed. The use of a four-electrodes measurement system decreased the dependency on the electrode interface’s impedance, and thus resulted in a systemmore sensitive to the cell features. The EISmonitoring of different cultures expansion showed the expected inverse proportional relationship between the central relaxation frequency and the cell cross-sectional area. The morphological changes of fibroblast cells during the detachment processes were also studied. Interestingly, EIS displayed the proportional relationship between the ¿ parameter of the Cole impedance model and the cell shape dispersion from sphericalmorphology (considering spherical shape ideality). CONCLUSION: The results obtained reveal the potential for developing a real-time monitoring tool for cell morphology features such as cell size and shape, which are involved inmany cellular processes like cell expansion, differentiation, cell attachment or cell death.Peer Reviewe

Development of a simple disposable six minibioreactor system for suspension mammalian cell culture

A simple disposable six minibioreactor system has been developed in order to perform multiple cell culture experiments in parallel, as a tool to accelerate experimentation in cell culture optimization. The system consists of a fixed part containing all instrumentation, sensors and actuators, and a disposable part, a compact unit with six minibioreactors with 10–15 mL of working volume each. This single-use unit is made of transparent biocompatible plastic material (polystyrene). Each one of the minibioreactors is equipped with agitation, headspace aeration supply and two optical probes, one for total cells measurement and pH, and another for dissolved oxygen measurement (and consequently the evaluation of Oxygen Uptake Rate, OUR). As an example of application, the performance of the system is successfully demonstrated for the culture of hybridoma cells growing in suspension under different conditions. The results allowed confirming the reproducibility of the system, and the feasibility to follow-up continuously the differences in cell growth, pH, pO2 and OUR evolution when hybridoma cells are cultured in different experimental conditions. For this, three different sets of experiments are considered. First, the use of the same culture medium DMEM supplemented with 10% fetal calf serum in all six minibioreactors. Second, the use of DMEM supplemented with three different FCS percentages (0, 1 and 10%) in two minibioreactors each. Third, the use of two different media (DMEM supplemented with 10% FCS and a chemically defined medium) in three minibioreactors each.Peer Reviewe

A simplified implementation of the stationary liquid mass balance method for on‐line OUR monitoring in animal cell cultures

BACKGROUND Compared with other methods, the stationary liquid mass balance method for oxygen uptake rate (OUR) determination offers advantages in terms of estimation accuracy and reduction of stress. However, the need for sophisticated instrumentation, like mass flow controllers and gas analysers, has historically limited wider implementation of such a method. In this paper, a new simplified method based on inexpensive valves for the continuous estimation of OUR in animal cell cultures is evaluated. The determination of OUR values is based on accurate operation of the dissolved oxygen (DO) control loop and monitoring of its internal variables. RESULTS The method developed was tested empirically in 2 L bioreactor HEK293 batch cultures. OUR profiles obtained by a dynamic method, global mass balance method and the developed simplified method were monitored and compared. The results show how OUR profile obtained with the proposed method better follows the off-line cell density determination. The OUR estimation frequency was also increased, improving the method capabilities and applications. The theoretical rationale of the method was extended to the sensitivity analysis which was analytically and numerically approached. CONCLUSIONS The results showed the proposed method to be not only cheap, but also a reliable alternative to monitor the metabolic activity in bioreactors in many biotechnological processes, being a useful tool for high cell density culture strategies implementation based on OUR monitoring