Hosted by the Biopharmaceutical Process and Quality Consortium (BPQC) - UMass Lowell and Tufts University, and the Massachusetts BioManufacturing Center (MBMC) of UMass Lowell

UMass Inn and Conference Center

Lowell, Massachusetts

Biopharmaceutical Summit 2012

FR IDAY, MARCH 9 , 2 012

bio project_Layout 1 2/28/12 9:06 AM Page 1

Dear Biopharmaceutical Summit participants,

Welcome to UMass Lowell – where we are building the future every day.

Over the past five years, we have taken on an ambitious acquisition and constructionschedule that is physically transforming our campus. Our new $70 million dollarEmerging Technologies and Innovation Center is on track for a fall 2012 completion.Construction has moved inside, where a buzz of activity surrounds workers buildingclean rooms and laboratories.

The new spaces will ensure that area companies in biomedical engineering and biotechnology, nanomedicineand other life sciences sectors can help guide our research so that our students are well prepared for the innovation economy.

But the progress is not all bricks and mortar. Our research expenditures have grown 64 percent in four years.A good portion of that research is industry-driven or industry-inspired.

The Massachusetts BioManufacturing Center at UMass Lowell (formerly the Bioprocess Development Center) was established here in 1993. It continues to serve area biopharmaceutical companies by innovatingtheir processes, conducting research and educating their employees. We help provide the workforce that keepsthe Massachusetts biotech supercluster strong.

The Biopharmaceutical Process and Quality Consortium–which organized today’s summit–will increase collaboration between academia and the biopharmaceutical industry, bringing both parties together to promote research innovation and commercialization.

With your help, UMass Lowell will keep building a promising future for your industry and the regional economy.

Sincerely,

Martin T. MeehanChancellor

Dear Colleague,

On behalf of the Massachusetts Life Sciences Center I would like to welcome you to UMass Lowell’s Biopharmaceutical Summit 2012! UMass Lowell is one of ourstate’s strongest contributors to innovative life sciences research and one of the manyreasons that Massachusetts is considered a global leader in the life sciences.

The Massachusetts Life Sciences Center is the agency charged with implementing thestate’s 10-year, $1 billion Life Sciences Initiative, a portfolio of investment tools thatwas proposed by Governor Deval Patrick, and approved by the Legislature in 2008.We work closely with the state’s world-class academic institutions, like UMass Lowell, and partner with industry and government to accelerate growth in the life sciences, and to make key investments that will both create jobs and support good science.

The Center has established a number of investment programs, including a Cooperative Research MatchingGrant Program that encourages sponsored research, a Life Sciences Accelerator Program that provides working capital to promising early-stage companies, and the Internship Challenge, a program that providespaid internship opportunities to students and recent college graduates at life sciences companies across Massachusetts. Our investments are playing an important role in making Massachusetts the world leader in life sciences innovation.

I hope that you will take a moment to visit our web site at www.masslifesciences.com to learn more.

Best,

Susan Windham-Bannister, Ph.D.President & CEOMassachusetts Life Sciences Center

Massachusetts Life Sciences Center, 1000 Winter Street, Suite 2900, Waltham, MA 02451, www.masslifesciences.com

Sponsors

TABLE OF CONTENTS

Schedule .......................................................................................................... 1

Presentation Abstracts ......................................................................................... 3

Panelists ........................................................................................................... 8

BPQC Members ................................................................................................ 14

Poster Presentations ......................................................................................... 16

1

Schedule

7:00–8:00 am Registration and breakfast

Welcome and Introduction

8:00–8:10 am Martin T. Meehan, Chancellor

8:10–8:20 am Dr. Julie Chen, Vice-Provost of Research UMass Lowell

Morning Session: Chair, Dr. Jack Prior, Senior Director of BioProcess Engineering, Enzyme

Plenary Presentations 1 and 2: Challenges and Innovations in Biopharmaceutical Industry

8:20–9:00 am Dr. Jorg Thommes, VP of Global Engineering, Biogen Idec: "Driving Value through BioPharmaceutical Manufacturing"

9:00–9:40 am Dr. William E. Bentley, Robert E. Fischell Distinguished Professor and Chair, Fischell Department of Bioengineering of the University of Maryland, College Park: "Translating Academic Research into Companies - Chesapeake PERL Reflections"

9:40–9:50 am Break

Invited Presentations: Experiences in Biopharmaceutical Manufacturing and Development

9:50–10:15 am Dr. Rajesh Beri, Director of MSAT, Lonza Biologics: "Implementation of Innovative Technologies and Best Practices in Biopharmaceutical Manufacturing"

10:15–10:40 am Dr. Brian Collins, Associate Director of Technical Operations, Momenta Pharmaceuticals: "Innovative Technology Implementation in Biopharmaceutical: Momenta Pharmaceutical Case"

10:40–11:05 am Dr. Cenk Undey, Director of Process Development, Amgen: "Experiences in Applied Advanced Process Analytics in Biopharmaceutical Manufacturing as an Innovative Technology"

11:05–11:30 am Dr. Bert Frohlich, Director of Bioengineering, Shire HGT: "The Future of Cell Culture Process Technology, Challenges and Advances"

11:30–12:10 pm Panel Discussion: Panelists: Dr. Thommes, Dr. Bentley, Dr. Beri, Dr. Undey, Dr. Frohlich, Mr. Steininger, Dr. Zhou and Dr. Clark

Networking Lunch: Chair, Dr. Kevin Bittorf, Associate Director of CMC, Vertex Pharmaceutical

12:10–12:40 pm Biopharmaceutical Process and Quality Consortium Introduction

Dr. Carl Lawton, Director of Mass Biomanufacturing Center, UMass Lowell Dr. Jin Xu, Co-Director of MBMC, UMass Lowell Dr. Seongkyu Yoon, Co-Director of MBMC, UMass Lowell Dr. Hyunmin Yi, Assistant Professor, Chemical and Biological Engineering, Tufts University Dr. Sanjeev Manohar, Director of Biomedical & Biotechnology Program, UMass Dr. Alex Fowler, Associate Provost of Graduate Studies, UMass Darmouth

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Plenary Presentation 3: Challenges and Innovations in Biopharmaceutical Industry

12:40–1:20 pm Mr. Bob Steininger, Head of Manufacturing/Operations, Acceleron Pharmaceutical: "An Alternative to the CMO Paradigm: Making Your Own with Single Use Technology"

Afternoon Session: Co-Chairs, Dr. Thomas Ryll, Senior Director of Cell-Culture Development, Biogen Idec, and Dr. Marty Sinacore, Director of Cell-Line Development, Biogen Idec

Plenary Presentation 4: Challenges and Innovations in Biopharmaceutical Industry

1:30–2:10 pm Dr. Michael J. Betenbaugh, Professor of Chemical and Biomolecular Engineering, Johns Hopkins University: "Chinese Hamster Ovary Genomics: Where Do We Go from Here?"

Invited Presentations: Innovation and Technology for the Biopharmaceutical Industry

2:10–2:35 pm Mr. Tom Fletcher, Director of Cell Culture R&D, Irvine Scientific: "Developing Better Culture Media for Producing Proteins in Fed-Batch CHO Culture Processes"

2:35–3:00 pm Dr. Mark Plavsic, Senior Director and Global Microbiological, Viral and TSE Advisor, Genzyme: "Contamination Control in Biopharmaceutical"

3:00–3:10 pm Break

3:10–3:35 pm Dr. Brian Lee, President and CEO, PBS Biotech: "A Novel Pneumatic Single-Use Bioreactor System for Flexible and Scalable Biomanufacturing"

3:35–4:00 pm Dr. Magdalena Leszczyniecka, CEO, STC Biologics: "Challenges in Biosimilar Development and Manufacturing"

4:00–4:25 pm Dr. Rahul Godawat, Process Scientist of Downstream Development and Dr. Tim Johnson, Manager of Process Development, Genzyme: "Platform Biopharmaceutical Process"

4:25–5:05 pm Panel Discussion

Panelists: Dr. Betenbaugh, Mr. Fletcher, Dr. Plavsic, Dr. Lee, Dr. Leszczyniecka, Dr. Godawat, Dr. Johnson, Mr. Makowiecki

5:05–5:10 pm Closing Remarks

5:10–7:00 pm Reception and Networking Poster Session

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Presentation Abstracts

8:20–9:00 am Driving Value through Biopharmaceutical Manufacturing

Dr. Jorg Thommes, VP of Global Engineering, Biogen Idec

Abstract

Over the last 30 years, the Biopharma Industry has had remarkable successes and has delivered tremendous value to our society through the development of innovative medicines. During this time our industry has also matured to an extent that the traditional paradigm of value creation solely through development and commercialization of innovative science may change. The economics of developing a drug pipeline became more challenging, the reimbursement environment is changing, biosimilars and increasing globalization add to the challenges. For the longest time, manufacturing was not part of the value equation. The transformation going on in our industry, however, requires that operations become part of the value chain. In this presentation we will discuss how biologics manufacturing can be turned from a necessary evil to a competitive advantage. We will show how focused innovation can further improve manufacturing efficiency and turn biotech manufacturing into a powerful value driver.

9:00–9:40 am Translating Academic Research into Companies - Chesapeake PERL Reflections

Dr. William E. Bentley, Robert E. Fischell Distinguished Professor and Chair, Fischell Department of Bioengineering of the University of Maryland, College Park

Abstract

Our research program has for many years focused on the development of innovative molecular tools that facilitate production of recombinant proteins in heterologous hosts. Tools such as feeding strategies, RNAi, transgene co-expression, etc., have been shown to increase yield and quality of proteins expressed in E. coli, yeast, insect cells, insect larvae and mammalian cell lines. Innovative concepts for increasing yield, lowering process development and production time that lead to linear scale-up processes were demonstrated, but in order to gain entre’ into the commercial sector required starting a company. Many of these revolved around the first demonstration over a decade ago that GFP could be used to monitor product expression – its use here was in insect larvae. The State of Maryland has several programs to facilitate technology start-ups – these and Federal programs provided significant leverage. Our discussion will touch on programs, capabilities, bottlenecks, as well as personal realization of strengths and weaknesses. Next generation innovations are also described.

9:50–10:15 am Implementation of Innovative Technologies and Best Practices in Biopharmaceutical Manufacturing

Dr. Rajesh Beri, Director of Manufacturing Sciences and Technology, Lonza Biologics

Abstract

Lonza Biologics facility in Portsmouth, NH began operating in 1996 with an installed capacity of 2,200 Liters. Over the past 15 years, the facility has seen significant growth in its size and scale of operation. The current production capacity at this site is 98,000 Liters. This talk will focus on the challenges and successes in implementing novel technologies and best in class procedures at this facility during its operating history.

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10:40–11:05 am Experiences in Applied Advanced Process Analytics in Biopharmaceutical Manufacturing as an Innovative Technology

Dr. Cenk Undey, Director of Process Development, Amgen

Abstract

Biopharmaceutical manufacturing processes are inherently complex due to their nonlinear bioprocess dynamics, variability in batch operations and manufacturing schedule, raw materials involved, and automatic process control. Incorporating innovative technologies into process development and manufacturing of a biological is critical to ensure improved process monitoring and control. Experiences in applying advanced process analytics in biopharmaceutical manufacturing environment and how this facilitates paradigm shift in operations will be summarized.

11:05–11:30 am The Future of Cell Culture Process Technology, Challenges and Advances

Dr. Bert Frohlich, Director of Bioengineering, Shire HGT

Abstract

Modern cell culture technology has certainly delivered on its potential from when it was born in the 1980’s. The number of biological drugs has risen steadily and the productivity of the cell culture processes that produce them has increased dramatically. For the foreseeable future, this technology’s future remains bright but there are significant challenges ahead. Biopharmaceutical manufacturing is maturing as an industry and will face the same pressures as other high-tech industries have undergone that preceded it. These pressures include rising healthcare costs, competition, and globalization. This talk will outline current trends and challenges that will impact cell culture technology as an applied science and manufacturing practice. The drive towards personalized medicine and a greater number of smaller-volume drugs will stimulate innovation in the related technologies of process intensification, sensors and automation, single-use (disposable) process equipment, and modular construction. Vaccines will continue to expand as more preventative medicine paradigms emerge and as markets expand into lesser-developed countries. Regenerative medicine will revitalize traditional cell culture methods. ‘Made-to-order’ specialty products requiring stem cell cultivation as well as tissue culture will take the technology even further in the direction of multiproduct manufacturing and reconfigurable manufacturing facilities.

12:40–1:20 pm An Alternative to the CMO Paradigm: Making Your Own with Single Use Technology

Mr. Bob Steininger, Head of Manufacturing/Operations, Acceleron Pharmaceutical

Abstract

Acceleron has made non-clinical and GMP material cost effectively in facilities based on a platform production process and single use technology for seven years. Over that time, a number of issues associated with the use of this technology have surfaced. In this talk, the advantages of using this technology and the challenges encountered will be reviewed. In addition, some of the industry’s recent success in standardization of at least one development step will be described, and used as a model approach to create more efficient production processes generally.

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1:30–2:10 pm Chinese Hamster Ovary Genomics: Where Do We Go from Here?

Dr. Michael J. Betenbaugh, Professor of Chemical and Biomolecular Engineering, Johns Hopkins University

Abstract

A new era is about to begin in the mammalian biotechnology community. Today, a quarter of all FDA approved new drugs are biopharmaceuticals, most of which are produced in Chinese hamster ovary (CHO) cells. With the sequencing of CHO K1 and other ‘omics efforts, we will now have access to much greater amounts of genomics, proteomics, metabolomics, and glycomics data. This presentation will discuss our previous work to sequence the genome of CHO cells and current efforts to organize, distribute, and share the rapidly expanding CHO genomics knowledge base in a framework that will benefit the entire community. We will also outline examples in which we are utilizing ‘omics data sets to alter mammalian cell performance. In one case study, we are evaluating the role of microRNAs in the activation and inhibition of the apoptosis cascade. Reaping the greatest benefits of this information explosion will require a new community infrastructure that will enable users to harness the ongoing ‘omics efforts in order to enhance performance of mammalian biotechnology systems.

2:10–2:35 pm Developing Better Culture Media for Producing Proteins in Fed-Batch CHO Culture Processes

Mr. Tom Fletcher, Director of Cell Culture R&D, Irvine Scientific

Abstract

As the biopharmaceutical industry matures, developing a competitive cell culture based protein production process becomes ever more challenging. With increased cost pressure, the advent of biosimilars and heightened expectations for process yield, comes a demand for more attention to every detail of the process. All of the major factors impacting the performance of a cell culture process, including 1) cell line, 2) culture media and 3) process control parameters, must be optimized in order to deliver a competitive level of performance. Several case studies will be given that describe how culture media (both growth and feed) were optimized to meet certain process requirements. The discussion of these studies will also explore what general lessons about cell culture behavior can be learned from them.

2:35–3:00 pm Contamination Control in Biopharmaceutical

Dr. Mark Plavsic, Sr Director and Global Microbiological, Viral & TSE Advisor, Genzyme

Abstract

Manufacture of therapeutics and vaccines in biological systems is associated with the potential risk of process and product contamination by adventitious viruses and other microbial agents. Although the source of virus and ways of its entry could vary, the use of raw materials of animal origin in the manufacturing processes is regarded as the most likely way of entry. Manufacturers typically control for this risk by careful raw material selection, vendor qualification, raw material and starting material (cell bank) testing, raw material treatment for viral clearance, facility design, manufacturing process design with built-in viral clearance steps, and risk assessment. We will review the viral risk management from a systematic and holistic perspective, and discuss the benefits of gamma irradiation as a means to mitigating the risk of raw material contamination by adventitious agents.

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3:10–3:35 pm A Novel Pneumatic Single-Use Bioreactor System for Flexible and Scalable Biomanufacturing

Dr. Brian Lee, President and CEO, PBS Biotech

Abstract

One of the challenges of cell culture process scale-up is to maintain a consistent physical and chemical environment in the bioreactors as vessel size increases. A pneumatic mixing mechanism has been developed using the Air-Wheel™ technology which converts the buoyancy of sparge gas bubbles into rotational energy, achieving efficient liquid and gas mixing without any external mixing device. The mixing system is scalable from 2L to 5,000L with fast mixing and high mass transfer rates (kLa >20 hr-1). In addition, average level of shear stress of the pneumatic mixing system (<0.3 Pa) calculated from computational fluid dynamics (CFD) modeling is significantly lower than in conventional stirred tank bioreactors (1.0–2.4 Pa) and remains constant during the scale-up. These results indicate that the cell culture environment in pneumatic mixing bioreactors is consistent across scales, which will make the process scale up from laboratory to production stage much more predictable and reliable. Cell culture performance of the pneumatic single-use bioreactor system (PBS) was evaluated using various cell types and processes including micro-carrier, perfusion, and high cell density fed-batch processes. In conclusion, PBS offers more homogenous mixing with lower shear stress than conventional stirred bioreactors and may offer improved performance in potential applications such as cell therapy, stem cell, regenerative medicine, viral production, and adherent cell culture on micro-carriers, in addition to traditional suspension cell culture processes for monoclonal antibodies and therapeutic proteins.

3:35–4:00 pm Challenges in Biosimilar Development and Manufacturing

Dr. Magdalena Leszczyniecka, CEO, STC Biologics

Abstract

Over the next few years, a global biosimilar market is likely to witness strong growth driven by the patent expiry of several major blockbuster monoclonal antibody drugs. Regardless of value, creating a biosimilar version of a biologic drug is not going to be as simple as creating a generic, simply because a complexity associated with producing biologics in living cells under a different process does not guarantee that the biosimilar version will display the same, or at least similar, product attributes. Establishing comparability of biologic drugs produced under a different process is not a trivial exercise. Currently, a wide variety of methods are used to characterize the structural diversity of post-translational profiles on drugs produced under different processes. Despite the advances in the field, current analytical methods are incapable of predicting which structural elements present on biologic drugs impact their pharmacology, increasing the need for animal testing. As variations will always be present on different batches of biologics, in vitro methods that could rapidly predict the effect of different processes on the pharmacology of biologic drugs would provide a significant advancement in the field of biologics manufacture.

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4:00–4:25 pm Building Better Biopharmaceutical Processes for the 21st Century – An Integrated & Continuous Biomanufacturing Platform

Dr. Rahul Godawat, Process Scientist of Downstream Development and Dr. Tim Johnson, Manager of Process Development, Genzyme

Abstract

The overarching business drivers for reducing development time and cost under stringent quality/regulatory requirements have dominated the biotech industry for many years, therefore, only incremental improvements have been adopted over time. As a result, the innovation in biotechnology has become more product than process oriented. Although these are still the dominant business drivers, other factors such as manufacturing flexibility capable of accommodating unstable/stable molecules, variations in production demands, and cost reduction are emerging as new drivers tipping the balance towards process innovation. In this study, we propose a universal, integrated and continuous biomanufacturing platform which addresses the conflicting business needs of speed, flexibility, and cost as well as quality. We have established a continuous biomanufacturing platform by integrating upstream and downstream operations for both relatively unstable (enzymes) and stable (antibodies) molecules. We will present data from this work where the cell-free harvest from a high cell density, steady-state, perfusion cell culture process was directly and continuously loaded onto a capture operation for extended time periods. The capture was based on periodic continuous chromatography (PCC) where product breakthrough from one column can be loaded on to next column resulting in near complete resin capacity utilization. Up to 70% increase in the utilization of resin capacity and corresponding 70% decrease in buffer usage was observed compared to the batch mode chromatography. The consistency of continuous capture with respect to in-process indicators and controls, and CQA’s was demonstrated for up to 640 column operations, 160 cycles, and 30 days. The streamlined process results in a significantly simplified process that is capable of reducing the facility foot print - 10 fold smaller bioreactor, >90 fold smaller capture column, elimination of non-value added unit operations such as hold tanks, clarification feed tanks, and clarification step, to name a few. Overall, the integrated bioreactor and capture system results in low costs, high efficiency, high volumetric productivity, and manufacturing flexibility. Based on these attributes, integrated continuous bioprocessing is being explored as a universal bioprocessing platform for the production of various therapeutic proteins at Genzyme.

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Panelists

Jack is the Sr. Director of BioProcess Engineering at Genzyme. His group supports data trending systems that monitor online and offline data from all Genzyme biologics sites, and also has deep expertise in process modeling, characterization, debottlenecking, and scheduling. In this role Jack has led technical investigations into product comparability and comparability and the development of viral inactivation technology. From 2000 to 2006, Jack led the Manufacturing Technical Support organization for U.S. Biologics manufacturing, supporting cell culture, purification, and fill finish operations. In this role Jack’s group supported the ramp up of Cerezyme production at the site and the technology transfer of Fabrazyme and Myozyme into large scale manufacturing. As part of efforts to understand and reduce manufacturing variability, Jack led the implementation of

statistical process monitoring for all of Genzyme’s purification operations. Prior to joining Genzyme Jack received a Doctor of Science in Chemical Engineering from the Massachusetts Institute of Technolology and a B.S. in Chemical Engineering from the University of Connecticut.

Mark Trusheim is founder and President of Co-Bio Consulting, LLC and advisor to the University of Massachusetts, with which he first began to work on biomanufacturing when he was a Board Member and Interim President of the Massachusetts Biotechnology Council. Mark is a former Special Government Employee for the FDA’s Office of the Commissioner and currently holds appointments at the University of Massachusetts and at MIT. Co-Bio Consulting helps facilitate academic, government and industry consortia to grow Life Sciences economic clusters through collective action and individual organization success. Clients include established biopharma firms, start-up biotechs, universities and government agencies. As an entrepreneur, he founded and was the first President and CEO of the diagnostics company Cantata Laboratories. Prior to Cantata, Mark worked at Monsanto/Pharmacia, culminating his career there as Co-President and Chief Operating Officer of Cereon Genomics, LLC–a $500M collaboration with Millennium Pharmaceuticals. Mark holds degrees in Chemistry from Stanford

University and Management from MIT’s Sloan School of Management, where he also currently holds an appointment as a Visiting Scientist.

Dr. Thomas Ryll completed his Ph.D. in Biochemistry and Biotechnology in 1992 at the Technical University of Braunschweig, Germany. After a year of Postdoc work in Germany, he joined Genentech in South San Francisco. From 1993 to 2000 Thomas focused on Cell Culture Process Development for a number of recombinant proteins including Lenercept (TNFrIgG), GP120, TPO and Raptiva. In 2000 Thomas joined Abgenix as Senior Scientist and Associate Director, Cell Culture Development. His contributions included the building of a cell culture development department, the development of Abgenix’s proprietary expression system based on cell:cell fusion, and an efficient medium and fed-batch process format for antibody production. Other accomplishments included the support of design, construction and start up of a new Process Sciences and Manufacturing facility as well as project team leadership. Thomas joined Tanox in 2003 as Director and Senior Director of Process Development and member of the management team. Responsibilities included all activities from cell line generation, cell culture,

purification and formulation development and analytical support as well as project team leadership. His current role is Senior Director of Cell Culture Development at Biogen Idec. He joined Biogen Idec in 2006 and relocated to Cambridge, MA recently. Thomas scientific interests focus around combining biological and engineering aspects for maximizing process efficiencies and product quality.

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Dr. Marty Sinacore has over twenty years’ experience in biopharmaceutical development, specializing in bioprocess development and cell line development. Over the course of his career, Dr. Sinacore played a key scientific and strategic role at three Boston-area biotech companies: Karyon Technologies, Inc., Genetics Institute/Wyeth(Pfizer) and now BiogenIdec. Dr. Sinacore completed his doctoral dissertation in the Department of Biochemistry and Biophysics at the University of Connecticut and conducted post-doctoral research in the Department of Clinical Pharmacology at Stanford University. Currently, he holds the position of Director of Cell Culture Development and Cambridge Site Head in Biogen Idec.

Dr. Jorg Thommes is currently the Vice President of Global Engineering and Facilities at Biogen Idec. In his function, he oversees all aspects of engineering for all Biogen Idec manufacturing facilities worldwide, and he is responsible for Biogen Idec Facilities worldwide as well as for Environmental Health & Safety, Security, and Infrastructure and Capital Management. Dr. Thommes holds a Doctorate in Chemistry from University of Bonn, Germany (with a thesis in mammalian cell culture technology), and an advanced University Teaching Degree (Habilitation) in Biochemical Engineering from University of Dusseldorf, Germany. Previous positions include Vice President of BioPharmaceutical Development at Biogen Idec, Senior Director of Process Biochemistry at Biogen Idec and Associate Director of New Technologies at IDEC Pharmaceuticals. He has served on organizing committees and chaired sessions at a multitude of national and international meetings (American Chemical Society BIOT

Division, Recovery of Biological Products, Society of Biochemical Engineering, CAP, GAB, PDA).

William E. Bentley is the Robert E. Fischell Distinguished Professor of Engineering and founding Chair of the Fischell Department of Bioengineering. He is also appointed in the Department of Chemical and Biomolecular Engineering at the University of Maryland, College Park and the Institute for Bioscience and Biotechnology Research. Dr. Bentley received his undergraduate (BS, '82) and Master of Engineering degrees ('83) from Cornell University, and his Ph.D. ('89) from the University of Colorado, Boulder, all in Chemical Engineering. Dr. Bentley has focused his research on the development of molecular tools that facilitate the expression of biologically active proteins. Recent interests are on deciphering and manipulating signal transduction pathways, including those of bacterial communication networks, for altering cell phenotype. He has served on advisory committees and panels for the NIH, NSF, DOD, DOE, USDA, and several state agencies. He co-founded a protein manufacturing company, Chesapeake PERL. Dr. Bentley is a Fellow of the AAAS and AIMBE and is an elected member of the American Academy of Microbiology.

Rajesh Beri received his Bachelor of Technology in Chemical Engineering from the Indian Institute of Technology, Mumbai, India in 1987 and his Master and Doctorate degrees in Chemical Engineering from Worcester Polytechnic Institute, Worcester, MA. He has over 17 years of experience working in the biotech industry in process development, process scale up, manufacturing and facility/equipment design for over 30 biologics products derived from either mammalian or microbial cultures. He has previously worked at BioChem Pharma, Dow Chemicals, GlaxoSmithKline and Amgen. In his current role as Director, Manufacturing Sciences and Technology at Lonza Biopharmaceuticals, Portsmouth, NH, he manages a group of 50 scientists and

engineers responsible for process transfer and scale up, process characterization, manufacturing and regulatory support. His hobbies include hiking, biking and coaching youth basketball.

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Dr. Cenk Undey joined Process Development at Amgen in 2003 where he leads the Process and Systems Analysis group in Global Process Engineering group within Process & Product Engineering. He has introduced, led the development and implementation of multivariate data analysis and real-time multivariate statistical process monitoring technology in cGMP for use in manufacturing floor during his tenure at Amgen, resulting in a paradigm shift towards continued process verification and improved process understanding. Dr. Undey has industrial experience with PAT tools currently in use in bio/pharmaceuticals manufacturing and is on the steering committee of Pharmaceutical Process Analytics Roundtable. He published many

journal articles, book chapters and co-authored two books, Batch Fermentation: Modeling, Monitoring and Control, and PAT Applied in Biologics Manufacturing and Process Development: An Enabling Tool for Quality-by-Design. He holds bachelor’s, master’s and Ph.D. degrees all in Chemical Engineering from Istanbul University in Turkey.

Bert Frohlich is a Ph.D. Biochemical Engineer with 20+ years of experience in the biotechnology, pharmaceutical, and chemical industries in process/facility design and bioprocess development. He is currently Director of Bioengineering at Shire HGT developing cell culture-based processes for manufacture of recombinant proteins primarily for enzyme replacement therapies.

Mr. Steininger joined Acceleron in March 2007 as Senior Vice President, Manufacturing and Process Development. He is responsible for managing the process development, analytical development, manufacturing, facility, and validation personnel that are involved in making clinical trial material in Acceleron’s single-use-equipment-based manufacturing facility, and also served as a VP within the Millennium Product and Portfolio Management organization. Prior to joining Millennium, he held multiple roles at Genetics Institute. Mr. Steininger is a member of the advisory board for the Massachusetts chapter of ISPE, an advisory board member of the Chemical Engineering Department of the University of Massachusetts, Amherst, and on the Biotechnology Advisory Board for the RISTA program at the Cambridge High School. Mr. Steininger received a S.B. in chemistry from Massachusetts Institute of Technology and an M.S. in Chemical Engineering from the University of California, Berkeley.

Dr. Michael Betenbaugh is Professor of Chemical and Biomolecular Engineering at Johns Hopkins University. He received his Ph.D. in Chemical Engineering from the University of Delaware. He rose through the ranks to Full Professor and served as Chair of the Department, renamed Chemical and Biomolecular Engineering. He is a researcher in the area of cell and metabolic engineering for mammalian, insect, and algal eucaryotes specializing in protein expression, apoptosis, glycosylation, metabolism, cell cycle, and chaperones. He received the Young Investigator Award from National Science Foundation, the James Van Lanen Award from the American Chemical Society, and the Merck Cell Culture Engineering Award. He has presented the Biochemical Engineering Award Lecture at the American Institute of Chemical Engineering and the Bayer Award Lecture in Biochemical Engineering, and has been inducted into Sigma Xi Research Honor Society and the American Institute of Medical

and Biological Engineers. He was a founding member of the Society of Biological Engineering and has serves as an Associate Editor for the journal, Biotechnology and Bioengineering.

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Tom Fletcher is currently Director of Cell Culture R&D at Irvine Scientific. He has been involved in the development and manufacturing of serum-free culture media for large-scale biopharmaceutical production processes for over 20 years.

Dr. Brian Lee is the president and cofounder of PBS Biotech Inc. He graduated in Biology from Seoul National University, holds a M.Sc. in Bacteriology from the University of Wisconsin, and a PhD in Biochemistry from the Michigan State University. With over 18 years of experience in R&D and biopharmaceutical industry, Brian is involved in commercial bioprocess development, scale-up, characterization and technology transfer of various biomanufacturing processes for DNA vaccine, antibodies, recombinant therapeutic proteins, and natural products. In 2007, Brian left Amgen to become President of PBS Biotech, Inc. He is involved with the American Chemical Society, Society of Industrial Microbiology, and European Society of Animal

Cell Culture Technology, Amgen Bioprocessing Center, the Biotechnology Program at Moorpark College, the Green Cross Corporation in Korea.

Dr. Magdalena Leszczyniecka is President and CEO of STC Biologics, Inc. She started STC Biologics with Dr. Birck Wilson focusing on development of monoclonal antibody biosimilars. Magdalena brings over 12 years of experience in oncology, with 10 years of experience leading drug development programs in Oncology. Before STC Biologics, she worked in two venture capital firms, Flagship Ventures and Atlas Venture. She led the research on MM-121, an anti-ErbB3 antibody now being co-developed between Sanofi-Aventis and Merrimack Pharmaceuticals. She received her Ph.D. from a joined program between Columbia University and NYU and MBA from Babson College.

Dr. Timothy Johnson is a Process Development Senior Manager in the Commercial Cell Culture Development Department at Genzyme, A Sanofi Company, in Framingham, MA. He received a Ph.D. in Chemical Engineering from the University of Washington, Seattle, in 1999. Tim began his career advancing the field of microfluidics technology while at The National Institute of Standards and Technology (2000-2002), Tecan Group Ltd. developing ADME-tox applications (2002-2003), and finally at BioProcessors Corp. (2003-2007), developing small-scale, high-throughput, cell culture applications. Since 2007, at Genzyme Corp., Tim has been responsible for supporting, understanding, and improving the production of commercial cell culture processes, for bioreactor process development, optimization, scale-up, scale-down, and technology transfer, and for the development of new cell culture technology platforms002E.

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Dr. Rahul Godawat obtained his undergraduate degree in Chemical Engineering from IIT, India, and worked for 3 years in a chemical manufacturing plant before joining the PhD program at the Rensselaer Polytechnic Institute, NY, in 2004. His PhD work mainly focused on gaining fundamental understanding of biomolecular binding to various surfaces and tying the interfacial phenomena to properties of water molecules at the interface. After finishing his PhD, he went on to work in Genzyme’s purification development group. His current role at Genzyme includes development of new technologies for downstream processing, development of next generation processes and supporting critical manufacturing investigations. In addition to work, he likes to spend time with his family especially playing with his three year old daughter “Teesha”.

Dr. Weichang Zhou received his Ph.D. from the University of Hannover, Germany in 1989. He conducted postdoctoral research in several biochemical engineering areas at German DECHEMA-Institute, Swiss Federal Institute of Technology (ETH-Zurich) and University of Minnesota, USA. Between 1994 and 2002, Dr. Zhou worked at Merck Research Laboratories in its Bioprocess R&D department, lastly as Associate Director. His groups developed Merck’s licensed manufacturing process for the pentavalent bovine-human reassortant live, oral Rotavirus vaccine, RotaTeq® and a large-scale PER.C6TM cell growth. Dr. Zhou has published over 45 scientific papers and holds two patents. He has presented many papers, organized and chaired symposia in international conferences on topics related to bioprocess monitoring and control, bioreactor engineering, cell culture engineering, viral vaccines and vectors, monoclonal antibodies and others.

Dr. Dave Clark is currently Senior Director of Manufacturing Sciences and Engineering within Medimmune, with responsibilities for technology transfer and manufacturing support for the Frederick Manufacturing Center. Dave has experience in the pharmaceutical industry in the areas of vaccine, Mab, and small molecules process development and clinical manufacturing. His industrial experience has been at the following companies: J&J/Centocor, Wyeth Vaccines, Apollon (DNA vaccine company) and Life Technologies. Dave earned his PhD in Microbiology from Rutgers University and held a 2-year post-doctoral position at the Institute of Molecular Biology at the University of Zurich.

Joseph Makowiecki is currently the Manager of Downstream Process Development and Pilot Plant at Xcellerex. Joseph has been with Xcellerex since 2006 and is responsible for the day to day operations of the Downstream Process Development and Pilot Plant departments. His duties include external and internal downstream technology transfer, process development, scale-up, process optimization, design and execution of scale-down models and virus clearance studies utilizing single-use technologies. Prior to his role with Xcellerex, Joseph has worked for Glaxo SmithKline, ID Biomedical, Shire Biologics and Bayer in various roles of increasing responsibility within the

Manufacturing and Process Development/Pilot Plant departments. Over the past 18 years he has been involved with the downstream manufacture, process development, technical transfer and scale-up of multiple diagnostic, vaccine and bio therapeutic products from mammalian, microbial and insect expression systems. Joseph holds a BS degree in Biological Sciences from Assumption College.

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BPQC Members

Dr. Seongkyu Yoon is director of the Massachusetts BioManufacturing Center (MBMC), process system engineering and an assistant professor in the department of Chemical Engineering of the University of Massachusetts Lowell. His research area is Life Sciences Systems Engineering. Research covers Process Analytical Technology (PAT) and Quality by Design (QbD), Application of Design of Experiment (DoE) and MultiVariate Data Analysis (MVDA), supply chain management in biologics, and chemometrics in life sciences. Research aims at developing innovative systems technology with which one can improve drug development efficiency and manufacturing productivity, and developing innovative diagnostic systems and tools for selected diseases with chemometrics framework. He is currently developing system tools using a genomics and metabolic flux analysis approach to explain variability to productivity and

quality of CHO (Chinese Hamster Ovary) mammalian cell-culture product. Integration of medical devices with multivariate statistical method is also being explored to develop practical diagnostic tools.

Dr. Yoon completed his Ph. D. in Chemical Engineering from McMaster University (Hamilton, Canada) under Prof. John F. MacGregor’s supervision. Afterwards, he worked at Umetrics (Kinnelon, NJ) with Dr. Svante Wold and Nouna Kettaneh. He provided consulting and teaching on multivariate data analysis, experimental design, and batch analysis in various industries, pharmaceutical, biologics, semi-conductor, petrochemical, and financial. Before joining UMass Lowell, Dr. Yoon worked at Biogen Idec Biopharmaceutical Inc. as process analytics group leader of manufacturing sciences. He implemented MSPC (Multivariate Statistical Process Control) to all unit operations of both commercial and clinical manufacturing. This pioneering work significantly improved manufacturing robustness and clarity. The MSPC system is now considered as an industry standard which most biopharmaceutical manufacturers adapted as common manufacturing system. He also worked at Hyundai Petrochemical (now LG Chemistry) as a process engineer and implemented Advanced Process Control and Real-time Optimizer to ethylene manufacturing process in early 1990.

Dr. Hyunmin Yi is currently an Assistant Professor at the Department of Chemical and Biological Engineering of Tufts University. He received his B.S. in Chemical Technology and M.S. in Biochemical Engineering from Seoul National University, and Ph.D. in Chemical Engineering from the University of Maryland at College Park. He has published over 30 research articles in peer-reviewed journals such as Analytical Chemistry, Journal of Materials Chemistry, Langmuir, Nano Letters, Biotechnology and Bioengineering, and Lab-on-a-Chip.

He has extensive service activities for the biochemical engineering community as a panelist at many NSF grant proposal review panels, reviewer for over 20 journals, and has been chair for several sessions at ACS and AIChE National Meetings. He is currently the lead-PI on two NSF research grants. Professor Hyunmin Yi’s broad research interests are viral nanobiotechnology and biosensors. In the first area, his group utilizes genetically modified tobacco mosaic viruses (TMV) for readily controlled metal nanoparticle synthesis toward applications in environmental catalysis, organic synthesis and energy. In the

second area, soft-lithographic techniques are enlisted for robust fabrication of polymeric hydrogel microparticles toward rapid and reliable in-situ bioprocess monitoring. The overarching theme in both of these areas is to understand and exploit the selective and programmable properties of biological and biochemical materials and interactions in facile fabrication and assembly of multifunctional materials.

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Dr. Jin Xu is director of the Massachusetts BioManufacturing Center (MBMC) Protein Analysis and Characterization Laboratory and an assistant professor in the UMass Lowell Chemistry Department. He currently oversees and actively participates in protein structural/functional studies, protein product characterization and analytical development at MBMC. With his expertise in protein chemistry and biophysics, Dr. Xu also designs and conducts studies on the relationship between protein folding and protein productivity/quality. Before joining UMass Lowell, Dr. Xu received his Ph.D in Biochemistry from the University of North Texas. Afterwards, he worked as Senior Research Scientist and Principal Scientist at Wyeth Pharmaceuticals for over five years, before most recently establishing and leading the protein chemistry group at Percivia, LLC.

Dr. Carl W. Lawton is director of the Massachusetts BioManufacturing Center (MBMC) and Associate Professor in the Department of Chemical Engineering at UMass Lowell. As director of the MBMC, Dr. Lawton is responsible for overseeing the coordination and completion of process development client services including expression development, fermentation and cell culture development, downstream processing, process optimization and characterization. He works closely with companies on the verge of biopharmaceutical production to give them the opportunity to utilize the Center’s services to economically address staffing needs and learning curve constraints and to optimize time to market. Dr. Lawton creates and teaches customized training programs for biopharmaceutical manufacturing workforce as well as advising and teaching undergraduate and graduate students in the fields of chemical and biochemical engineering and others. He also is responsible for developing and maintaining an applied research program which focuses on

technological advances to improve the quality, cost and productivity of large-scale biomanufacturing production. Before joining UMass Lowell and creating the MBMC, Dr. Lawton was a bioengineering process consultant to companies on both the east and west US coasts and in Canada.

Dr. Sanjeev Manohar is an Associate Professor at the Department of Chemical Engineering at the University of Massachusetts Lowell. He holds Master’s degrees in Chemistry from the University of Madras and in Organic Chemistry from Southern Illinois University and a Ph.D. in Organic/Polymer Chemistry from the University of Pennsylvania. His research is based on the synthesis and characterization of nanostructured materials for energy storage and medical applications; optically transparent, conducting films of carbon nanotubes on flexible substrates with performance that can rival commercial indium-tin-oxide conducting coatings; chemical warfare agent sensing using carbon nanotube coatings on flexible substrates; photocapacitors and batteries from dye-sensitized solar cells using a completely new design strategy involving plant extracts, and nanocarbons; green chemistry approaches to polymer/metal catalysts for fuel cells; synthesis and characterization of conducting polymer nanotubes/fibers and composites with noble metals; and controlled and targeted drug delivery across the blood-brain-barrier for

treatment of Alzheimer's disease using nanoparticulate drug carriers.

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Poster Presentations

Effect of Excess Light Chain Expression on Antibody Productivity and Structural Integrity

Prachi Bhoskar and Jin Xu,

University of Massachusetts Lowell, Department of Chemistry, Lowell, MA

Brett Belongia and Robert Smith, EMD Millipore, Bedford, MA

Abstract

During the antibody folding process, light chain (LC) initially folds and facilitates the folding of heavy chain (HC) followed by complete antibody assembly. In this manner, LC functions as a chaperone by promoting heavy chain folding and IgG assembly. Therefore, excess LC could be beneficial for antibody production. Excess LC also minimizes unfolded heavy chain polypeptide accumulation. Thereby it reduces aggregation caused by HC accumulation. IgG-producing cell lines have often been observed producing excess LC. However, due to limited cellular resources for protein production, antibody productivity may declines with elevated free LC production. Moreover, since nascent LC contains an unpaired C-terminal cysteine that is generally oxidized prior to secretion, free LC production could alter the redox balance and thereby generate under-disulfide-bonded IgG. Unformed disulfide bonds may then generate partial antibody and aggregates. As free LC expression has the opposing consequences of antibody productivity and structural integrity, it is imperative for antibody manufacturers to determine the optimum level of free LC expression for cell line selection and cell culture optimization in order to obtain cell cultures with high product titer and superior quality.

In this project, reversed phase-HPLC was used to determine the LC: IgG expression ratio in cell culture media. With regards to antibody structure, we have focused on disulfide bond formation, partial antibody generation as well as antibody aggregation. By analyzing samples from various clones and cell culture conditions, we were able to correlate the levels of LC expression/oxidation and antibody productivity/structural integrity. These results demonstrate that free LC analysis could serve as a novel and potentially critical parameter for IgG cell line development and process optimization.

An Intrinsic Protein Promotes Amyloid Beta Aggregation

Tyler Carter, Nirmal Paliwal, Jeremiah Karanja and Jin Xu

University of Massachusetts Lowell, Department of Chemistry, Lowell, MA

Abstract

A predominant modern theory links Alzheimer’s disease (AD)-related neurotoxicity to early-stage amyloid beta (Aβ) oligomers rather than mature Aβ fibers and plaques. It is believed that the process of Aβ fibrillization dictates Aβ toxicity, and that in vivo cofactors which mediate the kinetics of Aβ aggregation play a critical role in AD pathogenesis. As a potential modulator of Aβ aggregation, the effects of an intrinsic protein on Aβ fibrillization were investigated. Time course thioflavin-T (ThT) fluorometry demonstrated that a fully-sulfated variant of this protein with platelet-type von Willebrand disease (Pt-vWD) mutations dramatically enhanced Aβ fibrillization. Subsequent ThT studies indicated that the Pt-vWD mutations instigated Aβ aggregation, implying an interaction similar to that between the protein and von Willebrand Factor (vWF). Bis-ANS fluorometry and far-UV circular dichroism showed that the protein reduced the duration of Aβ oligomerization. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) demonstrated that Aβ aggregates and low molecular mass Aβ respectively appear and disappear more rapidly in the presence of the protein, and that the latter co-aggregates with Aβ. Multiangle laser light scattering (MALLS) confirmed that Aβ aggregates observed on SEC were fibrillar in nature. A preliminary cell-based cytotoxicity assay suggested that the protein alleviated Aβ neurotoxicity. These results revealed a potential link between this intrinsic “cardiovascular” protein and AD pathology.

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Chemometrics based image processing and delineation for the intraoperative mapping of nonmelanoma skin cancer

Nicholas Trunfio1, Hae Woo Lee1, Seongkyu Yoon1, and Anna Yaroslavsky2

1Massachusetts Biomanufacturing Center, University of Massachusetts, Lowell 2Department of Physics and Applied Physics, University of Massachusetts, Lowell

Abstract

Nonmelanoma skin cancers are one of the most common human cancers and their occurrence has been increasing at a remarkable rate. In general, most of nonmelanoma skin cancers are treated by surgery but major challenge is in that it is typically difficult to localize tumor cells by visual inspection during the operation because of its poor contrast. Over the last decades, various optical imaging techniques have been developed to aid intraoperative, accurate, real-time and cost-effective inspection of skin cancers. Many reports have shown that this approach can be effectively used with providing real-time high contrast images of skin cancers. However, when these techniques are utilized in practical field, decision on excision margin should be still made by surgeon and, in some cases, it can be confounded with other healthy structures, such as hair follicles or sebaceous glands. To improve efficiency of real-time tumor delineation process, it is highly recommended to extract meaningful information from high-dimensional optical spectra and distinguish them into different classes (for instant, tumor versus healthy).

The spectral pattern of optical images obtained from tumors can be used to discriminate them from healthy tissues or assist localization of them if they have distinct patterns. Typically, spectra from optical imaging measurements have tremendous information about the biological state of samples and the availability of this information is highly suited for a multivariate approach of chemometrics. The main challenge in biomedical application of chemometrics is a construction of simple, robust and accurate classification models including the tasks of raw spectra preprocessing, feature selection, model validation, etc. In this study, delineation performance of nonmelanoma skin cancers as an intra-operative tool will be addressed by using the proposed chemometrics-based image processing approach. In the previous research, feasibility of combination of multimodal reflectance and fluorescence polarization imaging (RFPI) with spectroscopic analysis of the reflectance images was investigated for intraoperative delineation of basal cell carcinomas (BCCs) and the spectral responses including the optical densities as well as their wavelength derivatives were calculated for assessment of benign and malignant stained skin structures. This study will investigate if delineation performance can be improved by using chemometrics based image processing methods. In order to increase the accuracy of classifying tumors and other healthy structures, several chemometrics techniques will be tested, including orthogonal partial least squares-discriminant analysis (OPLS-DA), support vector machine (SVM), etc. with combination of novel feature selection methods.

The Characterization of Oligosaccharide Structures of a Fusion Glycoprotein Product

Brian Michaels, Rutwik Patel, Prachi Bhoskar, Tyler Carter, Jin Xu

University of Massachusetts Lowell, Department of Chemistry

Abstract

Glycoproteins are proteins with oligosaccharide chains attached to their polypeptide side chains. The glycans are attached as a result of co-translational or post-translational modifications. This process is termed as glycosylation, and plays an important role in protein folding, which in turn affects its structure and function. For protein therapeutics, the glycans likely have a direct impact on pharmacokinetics/pharmacodynamics, immunogenicity and clinical efficacy. Due to the complexity of protein synthesis and folding, batch-to-batch variation in glycan structure was often observed during biopharmaceutical manufacturing. Determining oligosaccharide structures is therefore a crucial component of biopharmaceutical characterization and analysis. The two specific aims for this project include: 1) using a combination of HPLC and mass spectrometry methods to separate and identify the charge isoforms related to glycosylation heterogeneity, and 2) quantifying sialic acid content in the fusion protein by HPLC.

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Transcriptional Investigation of Lactate Metabolism of CHO Cells

Omer Karpuz

University of Massachusetts Lowell, Department of Chemical Engineering, Lowell, MA

Abstract

Lactate is one of the important metabolites of CHO cell culture. Accumulation of lactate during the early period of culture processes causes decrease in product yield and quality as well. After the increased amount of lactate, the depletion follows. Even though there are promising recent findings, there is still no detailed explanations of the phenomena of lactate decrease after early accumulation. There has been many attempts to solve this issue: Low glucose containing mediums ( Zhang et al 2004); manipulation of sugar transport by feeding different type of sugars (Walschin and Hu, 2007) ; Reducing the expression of lactate dehydrogenase, LDH -A and pyruvate dehydrogenase (Zhou et al, 2011)…etc. Nowadays, advances in omic technologies allow scientist to have the ability of having a deeper understanding of complexity of cell culture phenomena. Since CHO genomic researches started later than other genomes, it will take some more years to complete the whole genome of CHO cell which will be very useful tool for exploring the genetic structure and changes during the culturing process. Except couple of new studies there is no transciptomic study to understand the lactate metabolism of the CHO cell which plays a crucial role. But there are some promising approaches that reached interesting results in terms of changing the lactate metabolism: 1) Utilizing expression of the anti-apoptosis genes (E1B-19K, Aven and XIAPΔ) which drastically change the lactate metabolism (Dorai et al. 2009); 2) Effect of LDH-C expression of lactate consumption after temperature shift that is believed to be the reason of late phase of high density cultures and longevity of the cell culture by reducing the lactate and base accumulation( Szperalski et al. 2011); 3) Effect of copper sulfate addition to CHO cell culture process which also significantly decreased the lactate accumulation; 4) Feeding lactate or pyruvate as a carbon source during certain stages which maintains low level of ammonia, low levels of pCO2, a desired level of lactate as well and promotes glutamine consumption (Li et al., 2012).

My goal of this study is to investigate the transcriptomic, gene level responses to these four modifications to find out the differentially expressed genes. The set of genes connected to lactate metabolism will be searched through the comparison of gene expressions of each method. DG44 and Freestyle CHO-S Cell lines will be cultures by utilizing these four modifications mentioned above. Affymetrix CHO microarrays will be used to identify the transcriptomic changes related to lactate metabolism. Further strategies such as optimizing the cell culture medium or new genetic modifications will be built on the extensive analysis of this study.

Batch quality control of glycation of monoclonal antibody in mammalian cell culture

Kenneth Umemba, Seongkyu Yoon*

*University of Massachusetts Lowell, Department of Chemical Engineering

Abstract

The use of Chinese Hamster Ovary (CHO) cells for large scale production of monoclonal antibodies (Mab) has been a very popular platform. CHO cell lines are simple and yield reliable high expression levels. CHO cells have strong similarities in characteristics to human mammalian cells. As result, the market demand for the use of CHO cell lines for the production of therapeutic Mabs has grown tremendously. Despite the substantial progresses that have been achieved in the production of therapeutic Mab through the application of recombinant bio-processing techniques, Mab still pose great safety and health risks as therapeutic active pharmaceutical ingredients. They are inherently prone to structural heterogeneity during production and scale-up. This work would examine the applicability of several spectroscopy technologies (NIR, Raman, Fluorescence, X-Ray and so on) to detect intra and extracellular cell characteristics in submerged CHO cell culture. Using these spectral information, metabolic activities would be analyzed online and in-situ for limiting critical informative patterns. Batch control strategy would be designed and deployed in fed batch culture to optimize and control targeted glycation of the harvest Mab product.

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The Role of Disulfide Bond Formation in GPIb-Fc Aggregation

Jing Wen, Tyler Carter, Prachi Bhoskar and Jin Xu

University of Massachusetts Lowell, Department of Chemistry, Lowell, MA

Abstract

Glycoprotein Ibα (GPIbα), the major component of the platelet membrane bound GPIb-IX-V receptor complex, plays a key role in platelet aggregation and thrombus formation by binding to von Willebrand factor (VWF) on exposed subendothelial collagen following vascular injury. GPIbα has seven cysteines with a free thiol group present at Cys65. This unpaired Cys65 residue is believed to be responsible for in vitro GPIbα aggregation. The GPIbα aggregation mechanism was studied using a recombinant GPIbα-Fc fusion protein (GPIb-Fc), with GPIbα as a control. Heat-stressed GPIb-Fc and aggregates were purified by SEC and analyzed by reducing and non-reducing SDS-PAGE. Native AUC and SEC-MALLS determined the molecular weight distribution of the aggregates. Intinsic tryptophan fluorescence and far-UV CD monitored for aggregation-induced GPIbα structural changes. SPR measured the impact of aggregation on GPIbα activity. GPIb-Fc was incubated in the presence of denaturing (guanidine) and alkylating (iodoacetimide) agents to determine the nature of aggregation, while a Cys65Ser mutant was used to ascertain the role of unpaired Cys65 in GPIb-Fc aggregation. TMR-maleimide thiol labeling and peptide mapping confirmed the GPIbα aggregation pathway. GPIb-Fc aggregates were SDS-stable and reducible, indicating that aggregation was caused by covalent disulfide bond formation. Aggregates ranged in size from dimer to large oligomers. Aggregation resulted in an altered GPIbα structure and inactivated GPIbα from in vitro VWF binding. Heat stressing increased covalent aggregation at higher pH and partially denaturing conditions, while the Cys65Ser mutant confirmed the role of unpaired Cys in initiating GPIb-Fc aggregation. The near-identical monomer:aggregate thiol labeling ratio suggested that GPIbα aggregation was caused by thiol-mediated disulfide shuffling. Peptide mapping of TMR-maleimide-labeled monomeric and aggregated GPIbα demonstrated that all three GPIbα disulfide bonds are susceptible to free Cys65 attack, and could therefore potentially form intermolecular disulfide bonds. GPIb-Fc aggregates are inactive, non-native isoforms resulting from unpaired Cys65 attack of extant disulfide bonds, which initiates disulfide bond shuffling. Future studies will elucidate the potential physiological role of this unique GPIbα aggregation mechanism in platelet activation and aggregation.

Fabrication of Chitosan-poly(ethylene glycol) hybrid hydrogel microparticles via Replica Molding for robust biosensing platforms

Sukwon Jung and Hyunmin Yi*

Department of Chemical and Biological Engineering, Tufts University, Medford, MA

*hyunmin.yi@tufts.edu, (TEL) 617-627-2195

Abstract

Rapid and reliable monitoring of physiological events for facile bioprocess control is an unmet challenge. We strive to address this issue through development of robust fabrication schemes for high capacity biosensing platforms that can be enlisted to capture direct variables such as mRNA transcription and protein expression. Specifically, a naturally derived polysaccharide chitosan has high content of primary amines, which offer important chemical functionalities for biofabrication (the use of biological materials and interactions for fabrication). While chitosan can provide functionality itself, its high viscosity, poor solubility in water and low mechanical strength due to its rigid crystalline structure have limited its utility in functional hydrogel fabrication. Meanwhile, replica molding (RM) offers simple, robust, and inexpensive procedure to allow for reliable duplication of simple shape-encoded particles. In this work, we utilized chitosan/PEG (polyethylene glycol) blended prepolymer solution to fabricate microparticles containing single stranded DNA molecules covalently coupled to the co-polymer backbones. The results show well-defined uniform particles, chemical reactivity, and conjugation of probe biomolecules via efficient conjugation chemistry under mild and readily controlled fabrication and conjugation conditions. We believe that the methods demonstrated in this presentation hold significant potential toward facile fabrication of high capacity biosensing platforms.

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Technology comparison for measuring metabolic components in mammalian cell culture processes

Andrew Bawn*, Andrew Downey*, Omer Karpuz*, Seongkyu Yoon*

*University of Massachusetts Lowell, Massachusetts Biomanufacturing Center

Abstract

Three common technologies were used to measure the metabolites from samples collected from two cell lines each with three shaker flasks and a small-scale bioreactor used for cultivation. For each flask and bioreactor, samples were taken daily during the 12 or 15 day span of each cell line; Freestyle CHO-S and CHO DG44, respectively. The technologies being utilized are a membrane based technology, HPLC, and a Cedex Bio supplied by Roche Diagnostics. The primary metabolites being measured are glucose, glutamine, glutamate, lactate, ammonia/ammonium, sodium, and potassium. Measurements were taken in triplicates in order to determine the precision of each instrument and to compare each technology when measuring similar components. Results show that the Cedex Bio measured compounds more precisely when compared to other technologies. Similar trends were observed with all technologies however consistent, reproducible measurements were seen only with the Cedex Bio.

Batch quality control of monoclonal antibody product (MAb) in mammalian cell-culture

Andrew Bawn*, Seongkyu Yoon*

*University of Massachusetts Lowell, Massachusetts Biomanufacturing Center

Abstract

An increase in the product quality attributes (PQA’s) from monoclonal antibodies (MAb’s) produced from mammalian cell culture processes, specifically using Chinese hamster ovary (CHO) cells, was achieved and monitored using off-line and on-line measurements. Increased antibody productivity and quality was a direct result of decreasing the cultivation temperature from 37°C to 33°C while maintaining glucose concentrations between 6 g/L and 1.5 g/L. Using non-reducing SDS-Page techniques and Ellman’s assay, investigation into whether or not partial antibodies were formed and the thiol content of the solution was measured. An indication of better PQA was determined to be a lower thiol content (moles of thiol/moles of protein) and a cleaner SDS-PAGE gel. Multi-way PLS was implemented with on-line spectroscopic data to directly predict off-line metabolite trends and indirectly predict PQA. PLS models were created to predict the product quality at the end of the cell culture and at intermediate stages.

Characterization of high molecular weight aggregates from a CHO-expressed ECD receptor-Fc fusion protein

James Strand1; Jin Xu2; Chi-Ting Huang1

1Acceleron Pharma, Cambridge, MA; 2University of Massachusetts Lowell

Abstract

Aggregation is one of the more common impurities observed in recombinantly produced biotherapeutics and has implications on productivity and purity. The mechanisms behind the aggregation of recombinant IgG protein therapeutics from mammalian expression systems have been extensively studied; however, less information is available on the mechanism of aggregation in recombinant Fc-fusion proteins. In an effort to better understand the mechanisms behind aggregation of glycosylated fusion proteins produced in CHO cells, we have examined the high molecular weight (HMW) forms of a model Fc-fusion protein present during production in cell culture media using a combination of analytical and biophysical techniques. We observed two different classes of HMW aggregates from ProA purified ECD-Fc fusion protein, one disulfide-linked, and the other mediated by hydrophobic interactions, each with different folds. Both populations display less processed glycan structures.

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Rapid Assessment of Raw Material Quality for Therapeutic Protein Production in Mammalian Cell Culture Using Data Fusion with Multiple Spectroscopic Measurements

Hae Woo Lee and Seongkyu Yoon*,

Department of Chemical Engineering, University of Massachusetts, Lowell, MA

Collaborator: SAFC

Abstract

In mammalian cell culture producing therapeutic proteins, one of the important challenges is the use of several complex raw materials whose compositional variability is relatively high and their influences on cell culture is poorly understood. Under these circumstances, application of spectroscopic techniques combined with chemometrics can provide fast, simple and non-destructive ways to evaluate raw material quality, leading to more consistent cell culture performance. In this study, a comprehensive data fusion strategy of combining multiple spectroscopic techniques is investigated for the prediction of raw material quality in mammalian cell culture.

To achieve this purpose, four different spectroscopic techniques of near-infrared, Raman, 2D fluorescence and X-ray fluorescence spectra were employed for comprehensive characterization of soy hydrolysates which are commonly used as supplements in culture media. First, the different spectra were compared separately in terms of their prediction capability. Then, ensemble partial least squares (EPLS) was further employed by combining all of these spectral datasets in order to produce more accurate estimation of raw material properties, and compared with other data fusion technique. The results showed that data fusion models based on EPLS always exhibit best prediction accuracy among all the models including the individual ones of single spectroscopy, demonstrating the synergetic effects of data fusion in characterizing the raw material quality.

Characterization of glycosylation in CHO cell culture using fluorescence spectroscopy

Karina Riojas and Hae Woo Lee; Advisor: Seongkyu Yoon

Department of Chemical Engineering, University of Massachusetts-Lowell

Abstract

Application of spectroscopic techniques has proved to be a powerful tool for advanced monitoring of mammalian cell culture, aiming at consistent therapeutic protein production. Since these techniques can provide fast, simple and non-invasive methods to obtain biochemical information about cell culture processes, it offers significant advantages over other traditional off-line analyzes. Among several spectroscopic techniques, 2-D fluorescence spectroscopy employing multiple excitation and emission wavelengths has high potential in characterizing various kinds of proteins, peptides, vitamins and amino acids in both intracellular and extracellular environments, whose variation can be direct indication of specific metabolic status or dynamics of mammalian cells. However, potential of 2-D fluorescence spectroscopy in bioprocess monitoring has not been fully explored so far due to its complexity in analyzing multi-way spectral datasets.

The objective of the proposed study is to evaluate the feasibility of fluorescence spectroscopy in monitoring two types of recombinant CHO cell lines (DG44 and CHO-s Free). For this, amino acids and product quality attributes (glycosylation and aggregation) will be estimated in an integrated manner by analyzing 2-D fluorescence spectra with combination of chemometrics tool, such as PCA and PARAFAC. Then, the prediction accuracy of these fluorescence-based regression models will be evaluated. This will provide a systematic way of retrieving biochemical information and can be further implemented for real-time monitoring of mammalian cell culture for improvement of the product quality consistency.

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Optical Fiber Sensors for Biomedical Applications

Xingwei Wang, University of Massachusetts Lowell, Department of Electrical and Computer Engineering

Abstract

Prof. Xingwei Wang’s group at the University of Massachusetts Lowell has developed different kinds of fiber optic sensors for various biomedical applications. For example, a tapered fiber-optic biosensor has been developed for potential applications in clinical, pharmaceutical, food safety, routine tests, patient home care, surgery and intensive care; a miniature pressure sensor with high sensitivity and fast response time has been developed for measurement of blood pressure inside the coronary arteries; a miniature temperature sensor was invented to monitor temperature changes inside the arteries during angioplasty. A novel mechanism of photo-acoustic generation on the optical fiber tip has been explored. Gold nanoparticles were synthesized on the fiber tip and were used as a medium transferring optical energy into acoustic waves. By integrating this generation module and an ultrasound receiving module on a single optical fiber, the world’s smallest duplex ultrasound probe can be realized for biomedical imaging within limited space.

High Capacity Cation Exchange Membranes

Dr. Carl W. Lawton, Department of Chemical Engineering, University of Massachusetts-Lowell

Abstract

Despite the many significant operational advantages associated with membrane chromatography over traditional column based chromatography, membrane based adsorbers have not found much acceptance in the industry in downstream purification of antibody based products. This is especially true for the applications where membrane adsorbers can be used in a bind and elute mode. The principal reason for this is the low capacity of these membrane adsorbers coupled with a constraint on the maximum available size for manufacturing operations. In this poster, we aim to present a case study of cation exchange membranes that successfully address the potential problem of low dynamic binding capacity. Experimental results will be presented showing the effectiveness of the membrane adsorber to capture IgG directly from a cell free harvest as well as use of the membrane adsorber as a downstream polishing step.

Application of Process Analytical Technology in Protein Purification: Use of Second & Fourth Derivative Ultra Violet (UV) for Making Real-Time Pooling Decisions in Chromatography

Mark-Henry Kamga, Haewoo Lee, Seongkyu Yoon

Department of Chemical Engineering, University of Massachusetts Lowel

Abstract

Implementation of effective Process Analytical Technology (PAT) in biopharmaceutical operations has the potential to ameliorate operational compliance by facilitating and improving real time monitoring and control both in process development and manufacturing. These combined effects have the potential to enhance cost reduction initiatives and mitigate risk. Step yields in chromatographic operations are typically calculated using offline HPLC and data is usually available after completion making real-time control impossible. On-line monitoring has been effectively used to facilitate real-time pooling decisions. However, HPLC operations take averagely thirty minutes to complete, thus process control decisions are difficult to make during process runs which typically take one hour.This paper presents an alternative approach to estimating the protein concentration of effluent mixtures from a chromatographic operation using higher derivative of UV spectra by a Partial Least Squares (PLS) regression model. These spectra take less than thirty seconds to collect, making feedback control practical during process runs. The model predictions are compared to actual concentrations and HPLC analysis to estimate relative errors.

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Systematic media optimization of CHO cells for biosimilar development

Sumit Sekhar Dutta; Advisor: Seongkyu Yoon

Department of Chemical Engineering, University of Massachusetts-Lowell

Abstract

Traditional methods of media development are time consuming and inefficient in terms of time to be spent and information to be collected. Moreover, this approach does not explain the interdependency of two or more variables on each other. Due to the high cost of media, there is no denying the need for developing a chemically defined media which will cut the cost of culturing by a considerable amount. However, the attempt is to minimize the number of experiments and analyze as many variables as possible by a screening design. Following which, the non-critical elements are eliminated out and the critical elements are analyzed with a more detailed approach. The DG44 cell line will be used for this study. The DG44 CHO cells being an expressing cell line, the titer (amount of IgG production), VCD (cells/ml), glycosylation and the viability (%) will be the criteria for deciding the optimum concentration of each of the components to be tested. The metabolite concentration will be confirmed using Cedex Bio (Roche) and NOVA 400, the cell count information will be taken using Cedex HiRes (Roche) and Hemocytometer. Hydrolysates, glutamine, iron, HT supplement (Hypoxanthine and thymidine) are more important and will be addressed in detail in optimizing their concentrations. Moreover, I am hoping to look at the possibility of changes in media requirements, pre and post transfection of the DG44 cell line with GPF. This would enable me to get an idea about the effects of transfection on media requirements. This strategy will enable us to save a lot of time and resources and obtain an extensive data set, which will provide us the information on all media components on an individual level and in association and eventually reduce the development time for any biotherapeutics.