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Process Intensification of Spodoptera frugiperda (Sf) Cell Growth via Multi-Parallel Bioreactor System by Randy Stein B.S., Chemical Engineering University of Pittsburgh (2012) Submitted to the MIT Sloan School of Management and the Department of Chemical Engineering in Partial Fulfillment of the Requirements for the Degrees of Master of Business Administration and Master of Science in Chemical Engineering In conjunction with the Leaders for Global Operations Program at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY May 2020 © Randy Stein. All rights reserved. The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereafter created, Signature of Author _____________________________________________________________ MIT Sloan School of Management, Department of Chemical Engineering May 8, 2020 Certified by __________________________________________________________________ Kristala Prather, Thesis Supervisor Arthur D. Little Professor, MIT Department of Chemical Engineering Certified by __________________________________________________________________ Roy Welsch, Thesis Supervisor Professor of Statistics and Data Science, Sloan School of Management Approved by __________________________________________________________________ Maura Herson Assistant Dean of MBA Program, MIT Sloan School of Management Approved by __________________________________________________________________ Patrick Doyle Robert T. Haslam (1911) Professor of Chemical Engineering This Page is Intentionally Blank 2 Process Intensification of Spodoptera frugiperda (Sf) Cell Growth via Multi-Parallel Bioreactor System by Randy Stein Submitted to the MIT Sloan School of Management and the Department of Chemical Engineering on May 8, 2020 in Partial Fulfillment of the Requirements for the Degrees of Master of Business Administration and Master of Science in Chemical Engineering Abstract The objective of this project is to improve the yield of the fed-batch manufacturing process for the production of Flublok influenza vaccine, which was approved by the FDA in 2018. In short, Spodoptera frugiperda (SF+) insect cells are grown to a specific target cell density and then infected with baculovirus containing the gene of interest (GOI). For this particular process, the recombinant hemagglutinin (rHA) is extracted from the cell and used to produce the influenza vaccine. Protein Sciences developed a fed-batch process which improved on the traditional batch process by feeding supplementary nutrients to boost cell growth. The Fed-Batch process doubled the target cell density at the time of infection which resulted in a two-fold increase in the final yield of rHA and a 30% reduction in cost of goods. This Fed-Batch process can be further optimized to increase rHA yield and reduce the cost of goods. It is important to note that simply increasing cell biomass is not enough; the cells must also be able to produce rHA at a similar specific productivity in order to increase the yield. Exploratory process improvement experiments were performed on the ambr250 ® multi-parallel bioreactor system, with the goal of identifying the growth conditions for maximizing SF+ cell density. The conditions yielding the best results from these experiments were replicated in 3L glass bioreactors. Using data from these experiments, an optimized Fed-batch process can be developed. In addition, a statistical model was developed to relate key process parameters to SF+ cell density. This model can be used to quantitively ascertain how cell density is impacted by changing process parameters. Thesis Supervisor: Kristala Prather Arthur D. Little Professor, MIT Department of Chemical Engineering Thesis Supervisor: Roy Welsch Professor of Statistics and Data Science, Sloan School of Management Acknowledgements “Endings are never easy. I always build them up so much in my head they can’t possibly live up to my expectations, and I just end up disappointed. I guess it’s because we all want to believe that what we do is very important; that people hang onto our every word; that they care what we think. The truth is, you should consider yourself lucky if you even occasionally get to make someone, anyone, feel a little better. (Scrubs, My Finale)” This thesis is dedicated to my family, especially my mother and father, who helped me through some incredibly tough moments over the past two years. Right there with me were my Speakeasy friends, Houston friends, Lake Jackson friends, Sloanies, and roommates. Together, all of you formed a support network whose support and lightheartedness were immeasurable to me. For all their hard work, I would like to appreciate my advisors Roy Welsch, Kristala Prather, and Indresh Srivastava. I would like to include all of those in the LGO staff and faculty (especially Ted, Patty, Katja, and Anna) who spent numerous hours answering questions, resolving issues, and offering their support. Last, I would like to acknowledge the assistance that Mia from MIT Student Mental Health and Counseling gave me. Thank you to everyone at Sanofi Protein Sciences and Genzyme who made this work possible. I would like to acknowledge specifically the MSAT group at Genzyme, especially Alissa, Woo, and Canghai. Thank you to the great group of folks in Meriden: Nikolai, Jamal, Kate, Sam, Marcus, Liz, Mireli, and others. My gratitude also to Regis, Jean-Michel, and Sanofi- Pasteur for sponsoring the internship. I extend a final gratitude to Dana Cagle, Ryan Powell, and Johanna Graishe, for believing in me and writing the Letters of Recommendation that helped to get me in the program. I know you were not there to witness my work, but I hope that it lived up to your recommendations. I will certainly pay it forward. In addition, to all of the staff and operators at SPC: I always wanted to pass the lessons and knowledge I learned from you to classmates at Sloan. To the LGO class of 2020, congratulations and thank you for all the love and support! I hope our friendship continues to grow throughout the years. It seems like just yesterday I was driving more than thirty hours from Houston with Dante, and now it is all over. A special shoutout to fellow chemical engineers Ketan and Aaron, and teaching assistants Colin Grambow, Samuel Winslow, Thejas Wesley, and Joy Zeng, without whom I could not have passed Course 10. Finally, my Nonni and Nonno are always in my thoughts, who would have loved to see me finish a program like this. Also, to my dog Chip, who was a very good boy. And to you, to be honest, there is not a day that has gone by where I did not think about you. 4 Table of Contents Abstract ........................................................................................................................................... 3 Acknowledgements ......................................................................................................................... 4 Table of Contents ............................................................................................................................ 5 Table of Figures .............................................................................................................................. 9 Acronyms and Abbreviations ....................................................................................................... 11 1. Introduction ........................................................................................................................ 12 1.1 Thesis Outline ................................................................................................................ 13 2. Background ........................................................................................................................ 13 2.1 Impact of Influenza ........................................................................................................ 13 2.2 The Influenza Virus ........................................................................................................ 15 2.3 Protein Sciences ............................................................................................................. 16 2.4 Recombinant Flu Vaccines............................................................................................. 16 2.5 Batch Vaccine Production Process ................................................................................. 18 2.6 Fed-Batch Vaccine Production Process ......................................................................... 19 2.7 Project Motivation .......................................................................................................... 20 2.8 Project Overview ............................................................................................................ 21 3. Literature Review............................................................................................................... 23 3.1 Glucose ........................................................................................................................... 24 3.2 Glutamine and Glutamate............................................................................................... 26 3.3 Asparagine, Serine, and other Amino Acids .................................................................. 27 3.4 Ammonia, Lactate, and Alanine ..................................................................................... 28 3.5 Dissolved Oxygen and Oxygen Transport ....................................................................

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