Monoclonal Antibody Production and Purification

454 Pages·2017·22.24 MB·English

University of Pennsylvania
ScholarlyCommons
Department of Chemical & Biomolecular
Senior Design Reports (CBE)
Engineering
4-20-2018
Monoclonal Antibody Production and Purification
Hope E. McMahon
University of Pennsylvania, [email protected]
Jessica W. Schwartz
University of Pennsylvania, [email protected]
Shritama Ray
University of Pennsylvania, [email protected]
Follow this and additional works at:https://repository.upenn.edu/cbe_sdr
Part of theBiochemical and Biomolecular Engineering Commons
McMahon, Hope E.; Schwartz, Jessica W.; and Ray, Shritama, "Monoclonal Antibody Production and Purification" (2018).Senior
Design Reports (CBE). 104.
https://repository.upenn.edu/cbe_sdr/104
This paper is posted at ScholarlyCommons.https://repository.upenn.edu/cbe_sdr/104
For more information, please [email protected].
Monoclonal Antibody Production and Purification
Abstract
Monoclonal antibody (mAb) therapy is a form of immunotherapy that uses mAbs to bind mono-specifically
to certain cells or proteins. This may then stimulate the patient's immune system to attack those cells. MAbs
are currently used to treat medical conditions such as cancer, diabetes, arthritis, psoriasis, and Crohn’s Disease,
but have the potential to treat countless diseases and disorders. In 2015, the mAb market was valued at $85.4
billion, and is expected to reach $138.6 billion by 2024.1In manufacturing, mAbs are typically produced in
suspension in a series of fed-batch bioreactors using genetically engineered cells originally obtained from
Chinese Hamster Ovaries (CHO).2In this proposal, two upstream bioreactor designs were analyzed for
economic comparison given an annual production goal of 100 kg of mAb, with the first design culminating in
a 20,000 L volume at low mAb titer and the second design culminating with a 2,000 L volume at high mAb
titer. Following upstream mAb production, the protein was purified to meet clinical FDA standards using a
series of downstream purification techniques, including centrifugation, filtration, and chromatography. The
two designs can be modeled for both an on-patent and off-patent mAb in order to ensure long-term economic
viability. In this project, the drug was modeled based on Ocrevus (ocrelizumab), a humanized therapeutic
mAb brought to market in 2017 that targets a CD20-positive B cell to treat the symptoms of both primary
progressive and relapsing Multiple Sclerosis.3For an off-patent drug, it is recommended that the mAb be
priced at $35,000 per 1200 mg annual treatment in order to earn a 15% Internal Rate of Return (IRR) within
5 years of market uptake. For an on-patent drug, a price of $65,000 per 1200 mg treatment should be used to
recover the R&D costs of developing a new drug and sunk cost of past unsuccessful drugs. After analyzing
both designs, it was concluded that the second, smaller design scheme is more scalable, less risky, and more
cost effective for the production of both the on- and off-patent drugs.
Disciplines
Biochemical and Biomolecular Engineering | Chemical Engineering | Engineering
This working paper is available at ScholarlyCommons:https://repository.upenn.edu/cbe_sdr/104
Monoclonal Antibody
Production and Purification

Hope McMahon
Jessica Schwartz
Shritama Ray

Proposed by: Dr. Jeffrey Cohen
Project Advisor: Dr. John Crocker

University of Pennsylvania
School of Engineering
Department of Chemical and Biomolecular Engineering
April 17, 2018
April 17, 2018

Professor Bruce Vrana
University of Pennsylvania
Department of Chemical and Biomolecular Engineering
220 S. 33rd Street
Philadelphia, PA 19104

Dear Professor Vrana,

Enclosed are detailed process designs and economic analyses comparing two different bioreactor
design schemes for monoclonal antibody (mAb) drug substance production. The project proposal
specified an annual production goal of 100 kg mAb with a 90% process uptime. The goal of our
analysis was to design a process that would meet these requirements and recommend a selling
price that would provide a 15% internal rate of return (IRR). To more accurately model this
production project, we decided to base our design and economic decisions on a mAb that has
recently entered the market, Ocrevus (ocrelizumab). Using these parameters, we evaluated the
economic potential of our design schemes using both an on-patent and off-patent framework.

After careful consideration of the variables affecting drug quality and manufacturing
requirements, we designed two production schemes. The first upstream scheme culminates in a
20,000 L large production bioreactor, resulting in 25 kg of mAb per batch after downstream
purification. The second upstream scheme ends in a 2,000 L small production bioreactor to
produce 6.3 kg mAb per batch after downstream purification. Both designs are capable of
meeting the 100 kg annual production requirement within four to six months of the year. In order
to earn a 15% IRR within 4-6 years of production launch, we recommend pricing the off-patent
drug at $35,000 per 1200 mg annual treatment, and the on-patent drug at $65,000 per 1200 mg.
Considering both the economic and engineering analyses, we recommend implementing the
second, small bioreactor scheme, as it is more flexible, less risky, and more lucrative for the
production of both the on- and off-patent drugs.

Sincerely,

Hope McMahon Jessica Schwartz Shritama Ray
Table of Contents
1. Abstract 1
2. Introduction and Objective Time Chart 2
2.1 Project Background 2
2.2 Design Schemes 3
2.3 Ocrevus ™ 3
2.4 Objective Time Chart 4
2.5 Project Charter 5
3. Innovation Map 5
4. Market and Competitive Analyses 7
4.1 Competitive Landscape 7
4.2 Sales Forecast 7
5. Customer Requirements 8
6. Critical-to-Quality Variables 9
7. Product Concepts 11
8. Superior Product Concepts 12
9. Competitive Patent Analysis 13
10. Preliminary Process Synthesis 14
11. Assembly of Database 17
11.1 Chemical Components and Thermophysical properties 17
11.2 Cell Growth Kinetics and mAb Production Rate 17
12. Process Flow Diagram and Material Balances 19
12.1 Upstream Process for Large Production Bioreactor Design 19
12.1.1 Upstream PFD for Large Production Bioreactor Design (PFD 01) 20
12.1.2 Media Preparation Scheme for PFD 01 20
12.1.3 Overall Mass Balance for PFD 01 21
12.2 Downstream Process for Large Production Bioreactor Design 23
12.2.1 Downstream PFD for Large Production Bioreactor Design 23
12.2.2 Overall Mass Balance for PFD 02 23
12.3 Upstream Process for Small Production Bioreactor Design (PFD 03) 24
12.3.1 Upstream PFD for Small Production Bioreactor Design 24
12.3.2 Media Preparation Scheme for PFD 03 25
12.3.3 Overall Mass Balance for PFD 03 25
12.4 Downstream Process for Small Production Bioreactor (PFD 04) 26
12.4.1 Downstream PFD for Small Production Bioreactor Design 26
12.4.2 Overall Mass Balance for PFD 04 26
13. Process Description 27
13.1 Upstream Production Process Description 27
13.1.1 Media Preparation - Large & Small Production Bioreactor Processes 27
13.1.1.1 Growth Media 27
13.1.1.2 Feed Supplement Media 28
13.1.2 Operation Set Points and Controls 28
13.1.3 Inoculum Preparation - Large & Small Production Bioreactor Processes 31
13.1.3.1 250 mL Shaker Flask 32
13.1.3.2 25 L Single-Use Bioreactor 32
13.1.3 Bioreaction Processes - Large Production Bioreactor Process 33
13.1.3.1 100 L Single-Use Seed Bioreactor 33
13.1.3.2 500 L Single-Use Seed Bioreactor - Two in Parallel 33
13.1.3.3 2,000 L Single-Use Seed Bioreactor - Two in Parallel 34
13.1.3.4 20,000 L Stainless Steel Stirred Tank Production Bioreactor 35
13.1.4 Bioreaction Processes - Small Production Bioreactor Design 36
13.1.4.1 500 L Single-Use Seed Bioreactor 36
13.1.4.2 2,000 L Single-Use Production Bioreactor 37
13.2 Downstream Purification Process Description 37
13.2.1 Primary Recovery 38
13.2.1.1 Centrifugation 38
13.2.1.2 Depth Microfiltration 39
13.2.2 Initial Purification 39
13.2.2.1 Buffer Preparation 39
13.2.2.2 Protein A Chromatography 39
13.2.2.3 Virus Inactivation 41
13.2.2.4 Ultrafiltration / Diafiltration (UF/DF) 41
13.2.2.5 Cation Exchange Chromatography 42
13.2.3 Polishing Steps 43
13.2.3.1 Virus Filtration 43
13.2.3.2 Hydrophobic Interaction Chromatography 43
13.2.3.3 Ultrafiltration / Diafiltration (UF/DF) 44
13.2.3.4 Sterile Filtration 44
13.2.3.5 Final Storage 45
14. Energy Balance and Utility Requirements 46
15. Equipment List and Unit Descriptions 47
15.1 Upstream Production Process Units 47
15.1.1 Large Production Bioreactor Design 47
15.1.1.1 Blending Tank (PFD 01/P-1) 47
15.1.1.2 Dead-End Filter (PFD 01/P-3) 47
15.1.1.3 Storage/Blending Tank (PFD 01/P-5) 47
15.1.1.4 250 mL Shaker Flask with Orbital Shaker & Incubator (PFD 01/P-7) 48
15.1.1.5 25 L Single-Use Bioreactor (PFD 01/P-9) 48
15.1.1.6 100 L Single-Use Seed Bioreactor (PFD 01/P-29) 49
15.1.1.7 Blending Tanks (PFD 01/P-11; P-17; P-23) 50
15.1.1.8 Dead-End Filters (PFD 01/P-13; P-19; P-25) 50
15.1.1.9 Storage/Blending Tanks (PFD 01/P-15; P-21; P-27) 50
15.1.1.10 500 L Single-Use Seed Bioreactors (PFD 01/P-32,33) 51
15.1.1.11 2,000 L Single-Use Seed Bioreactors (PFD 01/P-36,37) 52
15.1.1.12 Blending Tank (PFD 01/P-46) 53
15.1.1.13 Dead-End Filters (PFD 01/P-48) 53
15.1.1.14 Storage/Blending Tanks (PFD 01/P-50; P-41; P-44) 53
15.1.1.15 20,000 L Stainless Steel Production Bioreactor (PFD 01/P-52) 53
15.1.2 Small Production Bioreactor Design 54
15.1.2.1 Blending Tank (PFD 03/P-1) 54
15.1.2.2 Dead-End Filter (PFD 03/P-3) 54
15.1.2.3 Storage/Blending Tank (PFD 03/P-5) 54
15.1.2.4 250 mL Shaker Flask with Orbital Shaker & Incubator (PFD 03/P-7) 54
15.1.2.5 25 L Single-Use Bioreactor (PFD 03/P-9) 54
15.1.2.6 Blending Tanks (PFD 03/P-11; P-17) 54
15.1.2.7 Dead-End Filters (PFD 03/P-13; P-19) 54
15.1.2.8 Storage/Blending Tanks (PFD 03/P-15; P-21) 54
15.1.2.9 500 L Single-Use Seed Bioreactor (PFD 03/P-23) 55
15.1.2.10 Blending Tank (PFD 03/P-31) 55
15.1.2.11 Dead-End Filter (PFD 03/P-33) 55
15.1.2.12 Storage/Blending Tanks (PFD 03/P-35; P-26; P-29) 55
15.1.2.13 2,000 L Single-Use Production Bioreactors (PFD 03/P-37) 55
15.2 Downstream Purification Process Units 55
15.2.1 Primary Recovery 56
15.2.1.1 Centrifuge (PFD 02/P-54, PFD 04/P-39) 56
15.2.1.2 Storage Tank 6 (PFD 02/P-56, PFD 04/P-41) 56
15.2.1.3 Depth Filtration Unit (PFD 02/P-58, PFD 04/P-43) 56
15.2.1.4 Storage Tank 7 (PFD 02/P-60, PFD 04/P-45) 57
15.2.2 Initial Purification 57
15.2.2.1 Protein A Chromatography Column (PFD 02/P-62 PFD 04/P-47) 57
15.2.2.2 Storage Tank 8 (Virus Inactivation) (PFD 02/P-64, PFD 04/P-49) 58
15.2.2.3 Ultrafiltration/Diafiltration (PFD 02/P-66, PFD 04/P-51) 58
15.2.2.4 Storage Tank 9 (PFD 02/P-68, PFD 04/P-53) 58
15.2.2.5 Cation Exchange Chromatography Column (PFD 02/P-70, PFD 04/P-55) 58
15.2.2.6 Storage Tank 10 (PFD 02/P-72, PFD 04/P-57) 59
15.2.3 Polishing Steps 59
15.2.3.1 Virus Filtration Unit (PFD 02/P-74, PFD 04/P-59) 59
15.2.3.2 Hydrophobic Interaction Chromatography Column (PFD 02/P-76, PFD 04/P-61) 60
15.2.3.3 Storage Tank 11 (PFD 02/P-78, PFD 04/P-63) 60
15.2.3.4 Ultrafiltration / Diafiltration Unit (PFD 02/P-80, PFD 04/P-65) 60
15.2.3.5 Sterile Filtration (PFD 02/P-82, PFD 04/P-67) 60
15.2.3.6 Final Storage (PFD 02/P-84, PFD 04/P-69) 61
15.3 Additional Units 61
15.3.1 Pumps and Tubing 61
15.3.2 Heating and Cooling 62
15.3.3 Cell Storage Tank 62
15.3.4 Biosafety Cabinet 63
15.3.5 Product Refrigeration Unit 63
15.3.6 Biowaste and Neutralization Holding Tank 63
15.3.7 Water for Injection Still 64
15.3.8 Sterile Air Compressor and Filtration 64
15.3.9 Clean Steam Generator 64
15.3.10 CIP Skids 64
15.3.11 Buffer Transfer Containers 65
15.3.12 Filter Integrity Test 65
15.3.13 Water Treatment Package 65
15.3.14 Biowaste Inactivation System 65
15.3.15 Waste Neutralization System 65
15.3.16 Quality Control Lab 66
16. Specification Sheets 67
17. Equipment Cost Summary 146
17.1 Large Production Bioreactor Design Equipment 146
17.2 Small Production Bioreactor Design Equipment 150
18. Scheduling 154
19. Fixed-Capital Investment Summary 157
20. Operating Cost- Cost of Manufacture 159
21. Profitability Analyses- Business Case 163
21.1 Off-Patent Analysis 164
21.1.1 Large Production Bioreactor Design 165
21.1.2 Small Production Bioreactor Design 165
21.1.3 Price Sensitivity Analysis 166
21.1.4 Sales Volume Sensitivity Analysis 168
21.2 On-Patent Analysis 169
21.2.1 Large Production Bioreactor Design 170
21.2.2 Small Production Bioreactor Design 171
21.2.3 Price Sensitivity Analysis 173
21.2.4 Sales Volume Sensitivity Analysis 174
21.3 Summary 176
22. Additional Considerations 176
22.1 Environmental Impact 176
22.2 FDA Regulations 177
23. Conclusions and Recommendations 179
24. Acknowledgements 180
25. References 180
Appendix A - Cryogenic Recovery of the CHO Cells 185
Appendix B - Growth Media and Feed Supplement Reconstitution 186
Appendix C - Clean-in-Place (CIP) & Steam-in-Place (SIP) Protocol 187
Appendix D - Oxygen Transfer and Gas Sparging 190
Appendix E - Exhaust Venting Rates 192
Appendix F - Economics 202
Appendix G - Vendor Specification Sheets 211
Appendix H - Material Safety Data Sheets 353
1. Abstract
Monoclonal antibody (mAb) therapy is a form of immunotherapy that uses mAbs to bind
mono-specifically to certain cells or proteins. This may then stimulate the patient's immune
system to attack those cells. MAbs are currently used to treat medical conditions such as cancer,
diabetes, arthritis, psoriasis, and Crohn’s Disease, but have the potential to treat countless
diseases and disorders. In 2015, the mAb market was valued at $85.4 billion, and is expected to
reach $138.6 billion by 2024.1 In manufacturing, mAbs are typically produced in suspension in a
series of fed-batch bioreactors using genetically engineered cells originally obtained from
Chinese Hamster Ovaries (CHO).2 In this proposal, two upstream bioreactor designs were
analyzed for economic comparison given an annual production goal of 100 kg of mAb, with the
first design culminating in a 20,000 L volume at low mAb titer and the second design
culminating with a 2,000 L volume at high mAb titer. Following upstream mAb production, the
protein was purified to meet clinical FDA standards using a series of downstream purification
techniques, including centrifugation, filtration, and chromatography. The two designs can be
modeled for both an on-patent and off-patent mAb in order to ensure long-term economic
viability. In this project, the drug was modeled based on Ocrevus (ocrelizumab), a humanized
therapeutic mAb brought to market in 2017 that targets a CD20-positive B cell to treat the
symptoms of both primary progressive and relapsing Multiple Sclerosis.3 For an off-patent drug,
it is recommended that the mAb be priced at $35,000 per 1200 mg annual treatment in order to
earn a 15% Internal Rate of Return (IRR) within 5 years of market uptake. For an on-patent drug,
a price of $65,000 per 1200 mg treatment should be used to recover the R&D costs of developing
a new drug and sunk cost of past unsuccessful drugs. After analyzing both designs, it was
concluded that the second, smaller design scheme is more scalable, less risky, and more cost

effective for the production of both the on- and off-patent drugs.

1