An ultra scale-down tool set for the predictive design of a membrane separation procedure for preparation of human cell therapies A thesis submitted to University College London for the degree of Doctor of Philosophy in Biochemical Engineering By María Fernanda Masri The Advanced Centre of Biochemical Engineering Department of Biochemical Engineering University College London September 2014 To my grandfather and my family. Gracias. ii Abstract Tools that allow cost-effective screening of the susceptibility of cell lines to operating conditions which may apply during full-scale processing are central to the rapid development of robust processes for cell-based therapies. In this study, an ultra scale-down (USD) device has been developed for the characterization of the response of human cell lines to membrane-based processing, using just a small quantity of cells that is often all that is available at the early discovery stage. Key operating conditions investigated were cross- membrane flow rate, cell age prior to processing and cell concentration (viscosity). The impact was evaluated by cell damage on completion of membrane processing as assessed by trypan blue exclusion and release of intracellular lactate dehydrogenase (LDH). Similar insight was gained from both methods and this allowed the extension of the use of the LDH measurements to examine cell damage as it occurs during processing by a combination of LDH appearance in the permeate and mass balancing of the overall operation. The main cell line studied was a clinically relevant human fibroblast. As expected, increased shear rates led to significant increases in rate and extent of cell damage. Cells aged (21°C hold for 24 hours) before processing led to a doubling of the extent of damage. Increased cell concentration from 1x106 to 100x106 cells mL-1 gave no change in the proportion of cells damaged. Preliminary studies showed that increased shear stress also led to morphological changes and the appearance of apoptotic cells post-processing. Two other human cell lines were also tested briefly for cell damage; a neural stem cell line and a prostate cancer cell line. These appear to be less robust than the fibroblasts with, for example ~0%, ~18% and ~42% damage being observed at the lowest shear stress (~44 Pa) conditions for fibroblasts, prostate cells and neural stem cells respectively. The effects of increasing shear rate, age of cells or concentration varied for each of the cell lines studied. Overall, this work suggests how membrane processing may be used for the recovery of human cells for therapy and how USD studies can speed the route to manufacture. iii Declaration I hereby declare that the work presented in this thesis is solely my own work and that to the best of my knowledge the work is original except where otherwise indicated by reference to other authors. Fernanda Masri August 2014 iv Acknowledgements This project was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and encompassed the collaboration of several industrial and academic groups around the UK; University College London and Onyvax Ltd and ReNeuron through the supply of cells for investigation. First and foremost, I would like to express my gratitude towards Prof. Mike Hoare. Your continual guidance and input over the years have been invaluable throughout my master and doctoral studies. I have learned incredible amounts from you and it really has been a true honour and a privilege to work under your supervision. You have provided me with so many opportunities and given me endless support that went beyond your obligations. For all of that Mike, I am eternally grateful. I would also like to thank Dr Ivan Wall, my co-supervisor at UCL, for providing direction and support when needed and sharing his knowledge with me. Thank you to Dr Kate Lawrence, my unofficial co-supervisor at UCL, you have been paramount to the success of this research. You have shared your wealth of knowledge and expertise always with a smile on your face (and most of the times with a cup of tea in your hand). I wish you both all the best for your future endeavours. A special thanks to my lab friends who really have made this an unforgettable 4 years: Chris Longster (aka Ginge karaoke king) for the periodic update on the Daily News; Kate Lawrence (aka Cake) for the endless supply of sweets to the office; Mike Delahaye (aka Yoda) for “training” me to face USD research during my masters and v beyond; JP Acosta (aka Mexi-CAN) for introducing me to cell-bioprocessing; Alex Chatel (aka Camaron de la playa) for (rather infrequent!) baking amazingness; and Jen Man (aka karaoke queen) for sharing my passion for musicals. Thank you to many other UCL members who make the department a special place, to name few Helmi, Sara, Bettsy, Hughson, Lourdes, Kate Pinto, Vish, Jayan, Rhys, Rooney, Doug, Daria, Matt, Asma, recent addition “Daaaave”… I could go on. Also thank you to my friends outside UCL who have accompanied me during this whole journey and have helped me define myself as a person; the original Poly water polo girls and coaches (all of them), the all of the Goodenough College crowd (but especially Z-dawg), my friends in Spain (Bacabarden) and the G-club. And Subhi, thanks to you, this thesis is a whole lot better! I know you have loved and enjoyed every minute of proof-reading. I truly thank you for your support, for your ص بحي ي ا أح بك أن ا .love and for your patience No sería un agradecimiento sin reconocer a mi familia; papá, mamá y Marce. A ustedes les quiero agradecer por tantas cosas que no sé ni por dónde empezar. Gracias por dedicar tiempo para escucharme, ayudarme a resolver los problemas y demostrar su preocupación por mí. Gracias por brindarme apoyo incondicional y por estar dispuestos a darlo todo para asegurar mi felicidad. En definitiva, gracias por ser como son. Mamá, Papá: Una buena familia comienza con un buen ejemplo de los padres y ustedes son el ejemplo más claro de amor y de que el esfuerzo y el trabajo duro nunca son en vano. Los amo a los tres, gracias por todo. vi Contents Chapter 1. Introduction ......................................................................................................... 14 1.1 Thesis overview .......................................................................................................... 14 1.2 Introduction to therapeutics ........................................................................................ 15 1.3 Cell-based therapies .................................................................................................... 16 1.3.1 Cell hierarchy ...................................................................................................... 19 1.3.2 Whole-cell cancer vaccines ................................................................................. 22 1.3.3 Stem cell therapies .............................................................................................. 23 1.3.4 Tissue engineering .............................................................................................. 25 1.4 Cell bioprocessing at full scale ................................................................................... 27 1.4.1 Upstream processing ........................................................................................... 30 1.4.1.1 Cell line selection ............................................................................................ 30 1.4.1.2 Cell culture and growth methods .................................................................... 32 1.4.1.2.1 Two-dimensional platforms ...................................................................... 32 1.4.1.2.2 Three-dimensional platforms .................................................................... 33 1.4.2 Downstream processing ...................................................................................... 35 1.4.2.1 Centrifugation ................................................................................................. 36 1.4.2.2 Continuous centrifugation ............................................................................... 37 1.4.2.3 Filtration .......................................................................................................... 38 1.4.2.3.1 Theory of flow through filters and transmission ....................................... 41 1.4.2.3.2 Bioprocessing stresses during TFF ........................................................... 43 1.4.3 Storage and delivery............................................................................................ 44 1 1.4.4 Scalability of cell-based therapies ....................................................................... 46 1.5 Ultra-scale down (USD) technologies ........................................................................ 48 1.5.1 USD membrane separation device ...................................................................... 49 1.6 Disruption of cell homeostasis: identification and assessment of cell quality attributes (CQAs) of the target cell population ...................................................................................... 50 1.6.1 Membrane integrity ............................................................................................. 55 1.6.1.1 Trypan blue exclusion dye as a measure of cell viability ............................... 56 1.6.1.2 Lactate dehydrogenase (LDH) release as a measure of cell death .................. 57 1.6.2 Protein expression for cell death ........................................................................