Multiparameter Sensor System for SingleUse Bioreactors Henry Lindner

Multiparameter Sensor System for Single-Use Bioreactors Henry Lindner and Konstantin Zouboulis in Analytical Strategy 2018 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Henry Lindner, Konstantin Zouboulis | 23. 10. 2018 | 1

Single-Use Bioreactors § Cultivation of cells or microorganisms § Cultivation tank made of multi-layer polymers § Size from several m. L up to 2000 L § Delivered ready to use 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 http: //technologyinscience. blogspot. com/2013/07/single-use-bioreactor-types-advantages. html | 23. 10. 2018 | 2

Single-Use Bioreactors Advantages Challenges and Perspectives § Increased facility flexibility § New sensor systems needed § Reduction of cross-contamination risks § No steam sterilization § Lower investment and energy costs § Transparent, thin polymer shell § Environmentally friendly § Single-use 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Henry Lindner, Konstantin Zouboulis | 23. 10. 2018 | 3

Relevant Biological Parameters for Cell Growth § External bioprocess conditions effect the cellular physiology p. H § Affects structures of proteins and macromolecules via protonation § Indicator for culture trajectory and contaminations p. O 2 § Cell respiration --> necessary for cell survival § Indicator for cell viability and culture trajectory Glucose § Primary carbon source for cell growth and metabolism Lactate § Primary waste metabolite --> toxic in high concentrations Ammonium § Minor waste metabolite --> toxic in high concentrations 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Henry Lindner, Konstantin Zouboulis | 23. 10. 2018 | 4

Importance of Proper Process Guiding § Controling growth and culture trajectory § Optimizing possible feeding strategies § Utilizing the full potential of the organisms § Ensuring high product quality, maximum yield and reproducebility § Detecting and preventing cell apoptosis, impaired products and batch loss 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Demuth, 2016 | 23. 10. 2018 | 5

Requirements for a Multiparameter Sensor System Process Conditions § Processes often last several days or weeks § Process temperature: 30 -40°C § Homogeneous batch (stirred) 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Sterilization and Calibration § Sterilization of the sensor with gammaradiation together with the bioreactor § Ready-to-use, pre-calibrated Henry Lindner, Konstantin Zouboulis | 23. 10. 2018 | 6

Requirements for a Multiparameter Sensor System Handling Economical Aspects § Disposable sensors or non-invasive interface § Cost-effective § Small § Easily producible in large amounts § Multi-sensor platform favorable § Pre-calibrated and installed by the § Modular architecture preferred manufacturer § Suitable sensor system for process 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Henry Lindner, Konstantin Zouboulis | 23. 10. 2018 | 7

Analytical Specifications § Accuracy § Measuring range § Sensitivity § Linearity § Selectivity § Invasiveness § Stability § Response time § Robustness § Inline § Reproducibility § Online § Verifiability, Integrability 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Henry Lindner, Konstantin Zouboulis | 23. 10. 2018 | 8

Overview of Measurement. Principles for Biological Parameters p. O 2 Electrochemical Opt(ochem)ical § Clark electrode p. H § p. H electrode § ISFET § Optode with fluorescent dye Others 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 § Optode with indicator dye § RFID Glucose Lactate Ammonium § Biosensor with § p. H electrode GOx LDH or LOx and CEM § ATR-FTIR § Raman § Enzymatic § Whole-cell thermistor sensor (E. coli) Biechele, 2011 + Bluma, 2015 | 23. 10. 2018 | 9

Sensor System – Optical Dissolved Oxygen Sensor (p. O 2) § Immobilized fluorescent dye § Dyes: metal organic complexes of Ru, Pd, or Pt 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Biechele, 2015 + https: //www. sigmaaldrich. com/catalog/product/sial/76886 | 23. 10. 2018 | 10

Sensor System – Optical Dissolved Oxygen Sensor (p. O 2) § Excitation of the dye with light source § Quenching in the presence of oxygen § Measuring principle: § Intensity (Stern-Volmer equation) § Lifetime (phase shift) 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Demuth, 2014 | 23. 10. 2018 | 11

Sensor System – Optical Dissolved Oxygen Sensor (p. O 2) BENEFITS § No oxygen consumption during measurement § Long shelf-life CHALLENGES § More expensive than Clark sensors § Longer response time than electrochemical solutions § Good performance at lower oxygen levels § Unknown strength of signal drift § Long-term stability due to solutions against § Leachables and extractables within dyes photobleaching § Separation of optical sensor spot possible 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 § Bad performance at high oxygen concentration Henry Lindner, Konstantin Zouboulis | 23. 10. 2018 | 12

Sensor System – p. H-Optode § Immobilized fluorescent indicator dyes § Dyes: Fluorescein or pyranine derivatives § Dual lifetime referencing for higher stability 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Demuth, 2016 + https: //www. sigmaaldrich. com/catalog/product/sial/56360 | 23. 10. 2018 | 13

Sensor System – p. H-Optode BENEFITS CHALLENGES § Flexible § Small measuring range § Cheap § Cross-sensitivity to ionic strength § Small in size § Photobleaching § No reference electrode required § Unknown strength of signal drift § No loss of sensitivity during sterilization § Leachables and extractables within dyes 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Henry Lindner, Konstantin Zouboulis | 23. 10. 2018 | 14

Working Principle and Geometry of an ISFET § Source and drain connected to current § Gate electrode electrically isolated from drain and source electrodes § Gate material: oxides like Si 3 N 4 or Ta 2 O 5 § Positive charge on the outside causes an electric potential and creates a channel § Change in the current between the source and drain electrode is proportional to the p. H 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Lee, 2009 + Mross, 2017 | 23. 10. 2018 | 15

Benefits and Challenges in Utilizing ISFETs BENEFITS CHALLENGES § Short response time § Gamma irradiation causes p. H shift § Easily miniaturized § Penetrating the bioreactor is necessary § Shelf-life of several months § Technically demanding coverage § Integration with measurement electronics § Reference electrode needed --> CMFETs § Separation of ISFET from reusable electrics 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Barbaro, 2006 + Abramova, 2009 + http: //microsens. ch/products/ISFET. htm | 23. 10. 2018 | 16

Working Principle of an Amperometric Biosensor § Biological detection component, signal transducer and signal conversion unit § Production of a current when a potential is applied between two electrodes § Enzymatic, catalytic oxidation of for example glucose into gluconolactone and H 2 O 2 § The rate of this electrochemical reduction depends on bulk oxygen concentration 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Tu, 2016 + http: //www 1. lsbu. ac. uk/water/enztech/amperometric. html | 23. 10. 2018 | 17

Benefits and Challenges of Amperometric Biosensors BENEFITS CHALLENGES § Fast response time § Cross-sensitivity to oxygen § Possibility of miniaturization § Requiring an additional reference sensor § High specificity § Drifting effects in long-term use § Versatilie § Leaching of used mediators § Ability to measure non-polar molecules § Enzyme isolation and purification is needed 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 http: //appliedbiosensors. com/biosensor-technology/ + http: //www. novabio. us/lactate-plus/ | 23. 10. 2018 | 18

Working Principle of an ATR-IR § Mid-infrared beam is totally internally reflected at the phase boundary § Reflectance creates an evanescent wave that extends a few microns into the sample § Evanescent wave is attenuated or altered by energy absorbtion from the sample § IR beam is passed to the detector 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Roychoudhury, 2006 + https: //commons. wikimedia. org/w/index. php? curid=1280591 | 23. 10. 2018 | 19

Benefits and Challenges in Using ATR-IR BENEFITS CHALLENGES § Usable in strongly absorbing media (water) § Further processing necessary § Several variables can be determined simultaneously in real time § Data sets to train the algorithms are required to extract the relevant process information § Non-invasive § Separation of the loop from fibre optics 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Mazarevica, 2004 + https: //artphotonics. com/product/fiber-optic-atr-loop-probe | 23. 10. 2018 | 20

Comparison Between Electrochemical and Optical Measurements Advantages Electrochemical Disadvantages § Well understood § Generally fragile § Industry standard for many years § Susceptible to agressive analytes § Low production cost § Reference electrode needed § Faster response time Optical § Ease of miniturisation § Susceptible to photobleaching or leaching § No separate reference electrode § Temperature dependant § Continuous measurments § Slower response time § Suited to mass-production 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 O’Mara, 2017 | 23. 10. 2018 | 21

Proposal of an Multiparameter Sensor System § Modular with electronic and optic parts § Multi-sensor chip with 7 mm edge length § Integratable into a standard PG 13. 5 housing § Multiple individual sensor elements § Internal comparability between sensor § Disposable and single-use-ready 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Mross, 2017 | 23. 10. 2018 | 22

Summary and Outlook § Sensor systems must fit to the requirements of single use bioreactors § Single-use reactors enable new possibilities § Little translation of the research into industry yet § Standardization must be faced Ø Real-time release of products via process validation 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Henry Lindner, Konstantin Zouboulis | 23. 10. 2018 | 23

References § § § § Abramova, N. ; Bratov, A. Photocurable polymers for ion selective field effect transistors. 20 years of applications. Sensors 2009, 9 (9), 7097– 7110. Barbaro, M. ; Bonfiglio, A. ; Raffo, L. A Charge-Modulated FET for Detection of Biomolecular Processes: Conception, Modeling, and Simulation. IEEE TRANSACTIONS ON ELECTRON DEVICES 2006, 53 (1), 158 -166. Biechele, P. ; Busse, C. ; Solle, D. ; Scheper, T. ; Reardon, K. Sensor Systems for Bioprocess Monitoring. Engineering in Life Sciences 2015, 15 (5), 469– 488. Bluma, A. ; Höpfner, T. ; Prediger, A. ; Glindkamp, A. ; Beutel, S. ; Scheper, T. Process Analytical Sensors and Image-Based Techniques for Single-Use Bioreactors. Engineering in Life Sciences 2011, 11 (6), 550– 553. Busse, C. ; Biechele, P. ; Vries, I. de; Reardon, K. F. ; Solle, D. ; Scheper, T. Sensors for Disposable Bioreactors. Engineering in Life Sciences 2017, 17 (8), 940– 952. Dekker, L. ; Polizzi, K. M. Sense and Sensitivity in Bioprocessing—detecting Cellular Metabolites with Biosensors. Current Opinion in Chemical Biology 2017, 40, 31– 36. Demuth, C. ; Varonier, J. ; Jossen, V. ; Eibl, R. ; Eibl, D. Novel Probes for p. H and Dissolved Oxygen Measurements in Cultivations from Millilitre to Benchtop Scale. Appl Microbiol Biotechnol 2016, 100 (9), 3853– 3863. Demuth, C. Chemische Sensoren in der Bioprozessanalytik. Chemie in unserer Zeit 2014, 48 (1), 60– 67. Lee, C. ; Kim, S. K. ; Kim, M. Ion-Sensitive Field-Effect Transistor for Biological Sensing. Sensors 2009, 9 (9), 7111 -7131. Mazarevica, G. ; Diewok, J. ; Baena, J. R. ; Rosenberg, E. ; Lendl, B. On-line fermentation monitoring by mid-infrared spectroscopy. Appl Spectrosc. 2004, 58 (7), 804 -10. Mross, S. ; Zimmermann, T. ; Winkin, N. ; Kraft, M. ; Vogt, H. Integrated Multi-Sensor System for Parallel in-Situ Monitoring of Cell Nutrients, Metabolites, Cell Density and p. H in Biotechnological Processes. Sensors and Actuators B: Chemical 2016, 236, 937– 946. O’Mara, P. ; Farrell, A. ; Bones, J. ; Twomey, K. Staying Alive! Sensors Used for Monitoring Cell Health in Bioreactors. Talanta 2018, 176, 130– 139. Roychoudhury, L. ; Harvey, L. M. ; Mc. Neil B. The potential of mid infrared spectroscopy (MIRS) for real time bioprocess monitoring. Analytica Chimica Acta 2006, 571 (2), 159 -166. Tu, D. ; He, Y. ; Rong, Y. ; Wang, Y. ; Li, G. Meas. Sci. Technol. 2016, 27, 045108. Wencel, D. ; Abel, T. ; Mc. Donagh, C. Optical Chemical p. H Sensors. Anal. Chem. 2014, 86 (1), 15– 29. 529 -0043 -01 L Analytical Strategy, ETH Zürich 2018 Henry Lindner, Konstantin Zouboulis | 23. 10. 2018 | 24