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DESIGN A PLANT
To manufacture 100000, 10ml fill vials
per annum at 10mg/ml strength of
RITUXIMAB
HOME PAPER SUBMISSION
BAC...
INDEX
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB
Sr.no Title Pg No.
1 Introduction ...
INDEX
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB
23 Safety ,Health and Environment ...
1. Introduction
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 1
1. Introduction.
...
1. Introduction
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 2
A Rituximab is a ...
1. Introduction
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 3
apoptosis, or pro...
1. Introduction
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 4
1.4. IMPACT
The i...
2. Executive Summary
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 5
2. Executiv...
2. Executive Summary
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 5
Gross Cost ...
3.Process Selection
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 7
3. Literatur...
3.Process Selection
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 8
3.3 Chinese ...
4.Process description.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 9
4. Process...
4.Process description.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 10
4.1. Medi...
4.Process description.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 11
▪ DPBS (D...
4.Process description.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 12
several w...
4.Process description.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 13
4.2. Medi...
4.Process description.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 14
3.5 mL of...
4.Process description.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 15
Then, aft...
4.Process description.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 16
a) Ultraf...
5. Kinetics and thermodynamics of the process
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITU...
5. Kinetics and thermodynamics of the process
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITU...
5. Kinetics and thermodynamics of the process
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITU...
6. Process Block Diagram
Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 20
Inocul...
7. Site Selection
Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 21
7. Site Selec...
7. Site Selection
Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 22
7.1.6 .Power:...
7. Site Selection
Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 23
7.3 Prosperit...
7. Site Selection
Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 24
Water/Effluen...
8. Material Balance
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 25
8. Material...
8. Material Balance
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 26
8.1. Biorea...
8. Material Balance
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 27
The size of...
8. Material Balance
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 28
Total cell ...
8. Material Balance
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 29
8.3. Materi...
8. Material Balance
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 30
Table8.5: A...
8. Material Balance
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 31
Table8.6: A...
8. Material Balance
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 32
Table8.8: A...
8. Material Balance
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 33
8.8. Vials ...
9. Energy balance
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 34
9. Energy bal...
9. Energy balance
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 35
9.1.3.Maintai...
10. Equipment Design
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 36
10. Equipme...
10. Equipment Design
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 37
10.1.4. Imp...
10. Equipment Design
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 38
For product...
10. Equipment Design
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 39
10.2.Protei...
10. Equipment Design
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 40
 Multiple ...
10. Equipment Design
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 41
10.3.1. Des...
10. Equipment Design
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 42
Tangential ...
10. Equipment Design
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 43
d) Continuo...
10. Equipment Design
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 44
9.2.2. Fina...
10. Equipment Design
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 45
Steam in pl...
11. Mechanical design Of Bioreactor
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page...
11. Mechanical design Of Bioreactor
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page...
11. Mechanical design Of Bioreactor
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page...
11. Mechanical design Of Bioreactor
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page...
11. Mechanical design Of Bioreactor
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page...
11. Mechanical design Of Bioreactor
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page...
11. Mechanical design Of Bioreactor
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page...
11. Mechanical design Of Bioreactor
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page...
11. Mechanical design Of Bioreactor
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page...
12. Equipment Sizing
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 55
12. Equipm...
12. Equipment Sizing
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 56
12.3.Produc...
12. Equipment Sizing
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 57
Table 12. 4...
12. Equipment Sizing
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 58
12.6. ION E...
12. Equipment Sizing
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 59
12.6.2. Cat...
12. Equipment Sizing
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 60
12.7.Virus ...
12. Equipment Sizing
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 61
12.9. Air C...
13. Instrumentation and Process control
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB...
13. Instrumentation and Process control
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB...
13. Instrumentation and Process control
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB...
13. Instrumentation and Process control
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB...
14. HAZOP Studies
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 66
14. Hazard an...
14. HAZOP Studies
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 67
The table sho...
14. HAZOP Studies
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 68
Impeller
Spee...
15.Batch Scheduling
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 69
15.Batch Sch...
15.Batch Scheduling
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 70
Cooling
Ferm...
16.MOC Selection
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 71
16. MOC Selecti...
16.MOC Selection
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 72
Based on the ab...
17. Plant Layout
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 73
17. Plant Layou...
17. Plant Layout
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 74
The equipment l...
18.Financial Analysis
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 75
18. Finan...
18.Financial Analysis
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 76
18.1.11 G...
18.Financial Analysis
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 77
The gross...
18.Financial Analysis
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 78
18.2.3 Es...
18.Financial Analysis
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 79
20. Estim...
18.Financial Analysis
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 80
Table 20....
19. Total Cost of Production
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 79
19...
19. Total Cost of Production
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 80
Th...
19. Total Cost of Production
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 81
19...
19. Total Cost of Production
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 82
We...
20. Estimation of Working Capital
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page ...
20. Estimation of Working Capital
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page ...
21. Estimation of Financial Expenses
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Pa...
21. Estimation of Financial Expenses
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Pa...
21. Estimation of Financial Expenses
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Pa...
21. Estimation of Financial Expenses
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Pa...
21. Estimation of Financial Expenses
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Pa...
21. Estimation of Financial Expenses
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Pa...
21. Estimation of Financial Expenses
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Pa...
21. Estimation of Financial Expenses
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Pa...
21. Estimation of Financial Expenses
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Pa...
21. Estimation of Financial Expenses
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Pa...
22. Storage, Utilities and Effluent treatment
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R IT...
RITUXIMAB PLANT DESIGN
RITUXIMAB PLANT DESIGN
RITUXIMAB PLANT DESIGN
RITUXIMAB PLANT DESIGN
RITUXIMAB PLANT DESIGN
RITUXIMAB PLANT DESIGN
RITUXIMAB PLANT DESIGN
RITUXIMAB PLANT DESIGN
RITUXIMAB PLANT DESIGN
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RITUXIMAB PLANT DESIGN

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RITUXIMAB PLANT DESIGN

  1. 1. DESIGN A PLANT To manufacture 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB HOME PAPER SUBMISSION BACHELOR OF CHEMICAL ENGINEERING BY ANIL V VIBHUTE INSTITUTE OF CHEMICAL TECHNOLOGY MATUNGA, MUMBAI - 400019 2013-2014 YG
  2. 2. INDEX Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Sr.no Title Pg No. 1 Introduction 1 2 Executive summary 5 3. Process selection 7 4. Process Description 9 5 Kinetics and Thermodynamics of Process 17 5.1 Kinetics 17 5.2 Thermodynamics 19 6 Block Diagram 20 7 Site Selection 21 8 Material Balance 25 9 Energy balance 34 10. Equipment Design 36 11 Mechanical design of F003 46 12 Equipment Sizing 55 13 Instrumentation and Process control 62 14 Hazard and Operability(HAZOP) Analysis 66 15 Batch Scheduling 69 16 MOC Selection 71 17 Plant Layout 73 18 Financial Analysis 75 19 Total Cost of Production 79 20 Estimation of Working Capital 83 21 Estimation of Financial Expenses 85 22 Storage, Utilities and Effluent Treatment 95
  3. 3. INDEX Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB 23 Safety ,Health and Environment 97 24 Conclusions 100 25 References 101 26 Appendix A 103
  4. 4. 1. Introduction Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 1 1. Introduction. This document is a project feasibility analysis for manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB. 1.1.Rituximab Finding a cure for cancer has always been a goal for many health care professionals. Many have tried, but few have made as much of a stride as Dr. Antonio Grillo-López. He, along with several colleagues, pioneered a new drug named rituximab that serves as the first FDA approved antibody to treat cancer. Depending on the severity of the cancer, this drug could either completely treat or extend the lifetime of the patient. This extraordinary feat had very humble beginnings.
  5. 5. 1. Introduction Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 2 A Rituximab is a genetically engineered chimeric murine/human monoclonal antibody consisting of a glycosylated IgG1 kappa immunoglobulin with murine light- and heavy-chain variable regions (Fab domain) and human kappa and gamma-1 constant regions (Fc domain). Directed against the CD20 antigen. Rituximab is produced by mammalian cell (Chinese Hamster Ovary) suspension culture in a nutrient medium containing the antibiotic gentamicin. Gentamicin is not detectable in the final product. Rituximab is a sterile, clear, colorless, preservative-free liquid concentrate for intravenous administration. Rituximab is supplied at a concentration of 10 mg/mL in either 100 mg/10 mL or 500 mg/50 mL single-use vials. The product is formulated in polysorbate 80 (0.7 mg/mL), sodium citrate dihydrate (7.35 mg/mL), sodium chloride (9 mg/mL) and Water for Injection. The pH is 6.5. 1.2.Mechanism of Action Rituximab kills cancerous B cells via three main mechanisms: complement-dependent cytoxicity (CDC), antibody-dependent cell-mediated cytoxcity (ADCC), and apoptosis. CDC occurs when a large group of plasma proteins (complement) work together to destroy invading pathogens and malignant cells. Antibody-antigen complexes, such as the one between rituximab and CD20, activate the complement. The complement protein C1 binds to the tail of the rituximab antibody in a “lock and key” fashion and starts a series of reactions that creates a membrane attack complex lining the B cell membrane and then creating a pore to allow the cellular contents to escape and eventually die. ADCC is a process where the antibody-antigen complex forms and then attracts other components of the immune system, including natural killer cells. The receptors on these cells recognize and bind to the tail of the rituximab antibody. The natural killer cells also carry granules filled with cytotoxic molecules. When the granules are released after the natural killer cells bind with rituximab, they penetrate the cellular membrane of the B cell and cause pores that facilitate the release of cellular content leading to the cell’s death. The granules can also destroy the cells by attacking the nucleus. The final mechanism is known as
  6. 6. 1. Introduction Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 3 apoptosis, or programmed cell death. Apoptosis is defined the death of cells that occurs as a normal and controlled part of an organism’s growth or development. When rituximab bonds to CD20 and forms the antibody receptor complex, it signals the cell to start the process. The cytoskeleton collapses upon itself, the nucleus condenses, and the DNA fragments into small pieces via enzymes. Membrane-bound vesicles are also shredded. At the end of apoptosis, the cell has effectively destroyed itself. It is still unclear, however, whether these mechanism act independently or in concert. Despite this, rituximab still proves to be an effective cure. 1.3.Properties Sr. No. Property Value 1 Molecular Formula C6416H9874N1688O1987S44 2 CAS number 174722-31-7 3 Chemical Family Proteins 4 Melting Point Not applicable 5 Color Clear ,colorless liquid 6 Appearance 7 Molecular Mass 143859.7 g/mol 8 Boiling Point(degrees C) 100 9 Optimal pH range 6.5 10 Binding affinity for CD20 antigen 8.0 nM 11 Solubility in Water Soluble
  7. 7. 1. Introduction Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 4 1.4. IMPACT The impact that rituximab had is unprecedented. It provides a cure for curable lymphomas such as diffused large cell lymphoma. The lifetime of patients with incurable lymphomas have been extended. Recent numbers further demonstrate the success of rituximab. It has been considered the top anticancer drug in the world since 2001. Sales in 2010 alone totaled $6.7 billion. Each year, around 50,000 lymphoma patients are cured. Since its introduction in 1997 until 2010, over two million patients have been treated. Prior to this discovery, there has been a long of stagnation in finding cures or ways to extend lifetimes. Hopefully the success of Dr. Antonio Grillo-López will inspire others to follow in his footsteps. He, himself, has said that he is neither a saint nor a magician. Geduld led to his success, something that can be replicated by any ordinary person with persistence and determination. 1.5. Manufacturers 1 Dr. Reddy’s Laboratories Ltd.(INDIA) 2 Genentech , South San Francisco.
  8. 8. 2. Executive Summary Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 5 2. Executive Summary Product: RITUXIMAB Installed Capacity: 100000 vials of 10 ml each with strength of 10 mg/ml. Project Type: Greenfield. Project Site: Raigad. State- Maharashtra Raw Material Requirements: Raw material Batch req ( kg) Rate (Rs/kg) Annual cost(Rs) Inoc. Media Sol 129 368 47472 serum free media 246 18000 4428000 H3PO4 6275 8.5 53337.5 NaOH 5892 15 88380 Pro. A reg buff 3174 10 31740 Pro.A elution 5317 9.2 48916.4 Pro A equil 11562 9.2 106370.4 ION eq buff 1730 11.2 19376 ION wash buff 1732 22 38104 IOX el buff 97 18.5 1794.5 Nacl(1M) 1065 22 23430 ammonium Sulfate 74 480 35520 polysorbate80 2 110 220 sodium citratet dihydrate 0.25 2500 625 Total Raw material cost 4923286
  9. 9. 2. Executive Summary Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 5 Gross Cost of Production: 1437 Lakh of product produced. Sales: Selling Price (Average): 18000000 Rs/kg of Rituximab Total Sales: 1800 Lakh/ annum. Financing: Total Fixed Capital Investment: 1302.2 lakhs Debt: Equity Ratio: 1.5:1. Term Loan: 808.45 lakhs @ 12 % per annum. Promoter’s contribution: 274.87 lakhs (51% of equity) Organizational participation: 80.84lakhs (15% of equity). Public Issue: 183.25 lakhs (34% of equity). Project Evaluation: Break-Even Capacity: 40.28% (= 59.6 % of installed capacity.) Payback Period: 3 year 7 months. Return on Investment: 65.41 % Weighted average cost of capital: 19.2%. (Desired return on equity = 30%) Profitability Index: 1.18 Internal Rate of Return: 52.76 %
  10. 10. 3.Process Selection Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 7 3. Literature Survey: The literature survey is an important part of selecting the process as well as gathering the necessary data about the process. A number of reactions and process are surveyed. The exhaustive literature survey done for the cell culture process for monoclonal antibody production is presented below: Large-scale production of monoclonal antibodies 3.1Escherchiai coli (microbial fermentation): has been most commonly used for production of antibody fragments such as Fabs that are utilized when Fc-mediated effector functions are not required or deleterious.39 Simmons et al.40 demonstrated that efficient secretion of heavy and light chains in a favorable ratio resulted in the high-level expression and assembly of full-length IgGs in the E. coli periplasm. The technology described offers a rapid and potentially inexpensive method for the production of full-length a glycosylated therapeutic antibodies that do not have ADCC functionality. Mazor et al.41 also showed that it was possible to obtain full- length antibodies from combinatorial libraries expressed in E. coli. The full-length secreted heavy and light chains assembled into a glycosylated IgGs that were captured by an Fc-binding protein located on the inner membrane. Flow cytometry was used after permeabilization of the membrane and attachment of the antibody to a fluorescent antigen. 3.2Aspergillus niger: has also been used for the production of mAbs or antibody fragments; Ward et al.42 used N-terminal fusion to glucoamylase for both heavy and light chains to express a full length IgG in this fungus. In addition, the use of cell-free protein synthesis for recombinant protein production is emerging as an important technology. Goerke and Swartz43 recently demonstrated the utility of the technology using E. coli cell extracts to produce a number of proteins, antibody fragments and vaccine fusion proteins, with correct folding and presence of disulfide bonds.
  11. 11. 3.Process Selection Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 8 3.3 Chinese Hamster Ovary: The rapid development of high-yielding and robust Manufacturing processes for monoclonal Antibodies is an area of significant focus in the biopharmaceutical landscape. Advances in mammalian cell culture have taken titers to beyond the 5 g/l mark. Since CHO cell and other continuously cultured cells have low efficiency in completely oxidizing glucose to CO2 and H2O, one by-product of cell culture process is lactate accumulation, which can cause acidification of culture medium and lead to high osmolarity and low viability due to the alkali added to control the medium pH. A significant amount of work30- 32 has been performed to reduce lactate accumulation; however, the usefulness of this approach may be very clone dependent. The increased cell culture productivity has shifted the attention of bioprocess development to operations downstream of the production bioreactor. This has rejuvenated interest in the use of non-chromatographic separation processes. Conclusion: Among the various process available three of them are studied and analysed by considering all the factor such as yield, conversion, cost of raw materials, complexity and availability of the reactant, energy consumption and hence route 3 i.e. synthesis of Rituximab using Chinese Hamster Ovary is selected as final process. 3.4 Justification for conclusion: 1) Advantage of this method over previous method is that this method provides easier way to produce Rituximab as raw material used such as CHO cell culture is abundantly available. 2) Conversion and selectivity of process is very high as compared to other processes. 3) The process is environmentally safe and there is no effluent disposal problem. 4) Most of the raw material required are easily available from Mumbai. So based on above justification I select production of Rituximab using CHO cell culture .
  12. 12. 4.Process description. Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 9 4. Process description The production of biological molecules can separated in to two major groups of processes: UPSTREAM and DOWNSTREAM a) Upstream processes: this process is mainly involved in the actual production of the molecule. Cells are genetically engineered to produce the peptide or protein of interest. This is done by modifying the genetic material, the DNA, of these cells so certain genes are expressed. After a predetermined time or number of cellular life cycles, the media containing the cell and protein products is then sent for downstream processing. This solution quite impure, as it contains much cellular debris, such as DNA, cellular membrane proteins, fragmented products and host cell proteins. b) Downstream processes: this mainly involved in the purification of the upstream feed and the further processing of the product. The numerous purification process available centrifugation and chromatography are important for our production. Once the product has been adequately purified by various techniques, it is known as drug substance, which is then further processed (fill, packed, labeled) to become the drug product that is ultimately distributed and sold.
  13. 13. 4.Process description. Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 10 4.1. Medium preparation: CD CHO medium is a protein-free, serum-free, chemically-defined medium optimized for the growth of Chinese hamster ovary (CHO) cells and expression of recombinant proteins in suspension culture. With a proven track record of quality for more than 10 years, CD CHO medium contains no proteins or peptide components of animal, plant, or synthetic origin, as well as no undefined lysates or hydro lysates. We import the CHO medium from Life Technologies. 4.1.1. DMEM and buffer preparation. a) Materials ▪ DMEM (Dulbecco's Modified Eagle Medium): Gibco-Brl #12800-017, 1 pack for 1 L Powder with high glucose with L-glutamine with pyridoxine hydrochloride with 110 mg/L sodium pyruvate without sodium bicarbonate ▪ Sodium bicarbonate (NaHCO3): sigma # S 7277, 500 g (cell culture grade) ▪ 3X Distilled Water (D.W.) ▪ 0.22 µm vacuum filter (vaccucap 90): Gelman science #pn 4622-filterling capacity up to 10 L CHRTGPH-3 Y-3 CHRTGPH-2CHRTGPH Reaction-1 Batch reactor Filtration Reaction-2 Reaction-3
  14. 14. 4.Process description. Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 11 ▪ DPBS (Dulbecco's PBS): sigma #D 5652, 10 bottles/pack, 1 bottle for 1 L ▪ Trypsin-EDTA (10X): sigma #T 4174, 100 ml ▪ FBS (Fetal Bovine Serum): JBI #S 001-01, 500 ml. b) FBS Serum supplies growth factors and nutrients. Serum requirement is dependent on cell type. Some cell lines have been 'trained' to survive in medium with low serum. So, must check how much serum amount your cells need before starting cell culture. These are the serum variables you must consider. - The percentage of serum: Most cells require 5-20% in the medium for good growth (all of our cell line require 10% FBS). - The types of serum: Some cells like horse serum or calf serum, but our system use fetal Bovine serum (FBS/FCS). - Heat-inactivate serum: Serum in subjected to heat to inactivate components such as complement. To heat-inactivate serum 1) Thaw the frozen serum (company supply FBS in -20℃) at 37℃ for 5-6 hours. 2) Incubate the thawed serum at 56-65℃ for 30 min (shake the bottle gently in every 10 min) 3) Aliquot the serum in 50 ml conical tube and then seal with parafilm. 4) Freeze and store. Thaw aliquots in a 37℃ water bath as needed. (Caution !!) Serum is very expensive. Always aliquot and freeze serum, and add it to medium just before use. Store unused portions of thawed aliquots in the refrigerator, where it will be fine for
  15. 15. 4.Process description. Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 12 several weeks…Please do not waste serum!!! c) Preparation of DMEM * History of culture media Complete media range in complexity from the relatively simple Eagle's MEM [Eagle, 1950] - Complex to Medium 199 [Morgan, 1950], CMRL 1066 [Paker, 1957], MB752/1 [Waymouth, 1959], RPMI 1640 [Moore, 1967], and Ham-F12 [Ham, 1965]. The complex media contain a large number of different amino acids, additional vitamins and Extra metabolites in F12 (optimizing by cloning) and in Dulbecco's modified Eagle's MEM (DMEM) [Dulbecco, 1959], DMEM-F12 [Barnes and Sato, 1980]. 1) Measure out 5% less 3X D.W. than desired total volume of media. 2) Add powdered media to D.W. at RT with gentle stirring (Do not over-stir or heat). 3) Rinse remained powder in the package with D.W. and add it to media. 4) Add 3.7 g of NaHCO3 to make 45mM concentration/L of media. 5) Add D.W. to desired volume. Stir until dissolved (Do not over mix). 6) The final pH of the media should be pH 7.4. Adjust pH of media to 0.2-0.3 below the Desired working pH (~ 6.95) using 1 N NaOH or 1 N HCl (generally HCl). Add slowly With stirring. The pH units will increase 0.1-0.3 upon filtration. After pH has been adjusted, Keep container closed with aluminum foil until media is filtered. 7) Sterilize immediately by using a Gelman vaccucap 90 through dispense in 500 ml bottles. 8) Store media at 4℃ in the dark until use. .
  16. 16. 4.Process description. Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 13 4.2. Media for Cell Culture We are now using washable and reusable glass bottles to prepare our media and our PBS solutions. The media bottles have tops which are compatible with the disposable sterile filters from VWR. These bottles should be washed with bleach and water (3x with bleach and 9x with water), then dried and autoclaved. After being autoclaved you can spray the outside with EtOH and bring into the laminar flow cabinet. I would recommend opening the bottle and placing the lid upwards on the bottom of the cabinet and turn on the UV light for 10 minutes just to be sure. 4.2.1 CHO K1 (-) Media This media is suitable for non-transfected CHO K1, or transiently transfected CHO cells which do not have any antibiotic resistance. To prepare the media will need the following: 1. 430 mL of DMEM 2. 50 mL of heat inactivated FBS 3. 5 mL of Pen/Strep 4. 5 mL of Non-essential amino acids 5. 10 mL of L-glutamine Mix all reagents together and run through a sterile filter. 4.2.2 CHO K1 (+) Media This media is suitable for CHO K1 cells which stably express fluorescent proteins and are resistant to geneticin. To prepare the media you will need the following: 1.400 mL of DMEM – Low Glucose 2.50 mL of FBS
  17. 17. 4.Process description. Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 14 3.5 mL of Pen/Strep 4.5 mL non-essential amino acids 5.10 mL of L-glutamine 6.0.235g of geneticin The geneticin is not immediately soluble in the DMEM. It is recommended that you add the geneticin first to the warm DMEM to increase the solubility and maximize the concentration of geneticin in the sample. 4.3. Bioreactor media composition The composition of the fermentation media is as follows: Table 4.1: Media Composition SR.NO. COMPONENT 1 Amino Acid(20) 2 Vitamin (9) 3 Organic Compound(8) 4 Inorganic Salts The pH of the media is maintained at 6.5 using NaOH dosing and the temperature is maintained at 37 0 C. 4.4 Chromatography: This is very common separation process that is used in many different industries. Small resin beads, typically agarose or polyacrylamide, contain surface properties that allow for the binding of specific molecules. These can either be molecules of interest, or the impurities that need to be removed. In this case of the former, a solution containing the protein of interest is pumped through resin, resulting in the protein binding to the resin. There are very specific condition, such as the pH and polarity of the solution, that allow this interaction to take place.
  18. 18. 4.Process description. Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 15 Then, after the impurities have washed through the resin column, another solvent, the eluent, is used to remove the proteins off the resin. Again, solution properties, such as the pH, are critical to allow for the dissociation of the protein from resin. This common modification known as a “bind and elute” mechanism, specific chromatography processes include the following: 4.4.1 Protein A Chromatography: This is a chromatography method that is mainly used when purifying monoclonal antibodies. In this case, the resin beads have many particles of proteins A attached to them. Protein A is unique in that it is able to bind mainly to the Fc( fragment, crystallizable ) portion of the monoclonal antibody with great specificity and potency. So , when a solution containing monoclonal Antibodies is passed through a column with protein A resin, the antibodies bind to the resin, while the impurities pass through. Resins that contain Protein A are very expensive, but they are effective as well. 4.4.2Cation Exchange Chromatography: In this method, the resin contain negative charges, anions so positively charged molecules are attracted to them. As the positively charged molecules attach to the resins, the more negatively charged molecules continue to flow through the column. It is conventional for the protein of interest to be bound to the resin, while impurities pass through. 4.4.3. Anion Exchange Chromatography: In contrast to cation exchange, the resin in this method are positively charge. Thus, they attract anionic (negatively charged ) molecules, allowing the positive ones to pass freely. 4.5. UF/DF: There are numerous type of filters that are utilized in the separation process of biologics. The step yields are generally pretty high >90% (less than 10% of the product is usually lost) common methods of filtration are following:
  19. 19. 4.Process description. Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 16 a) Ultrafiltration: This process is used to remove large molecules/impurities. The solution is forced through a semi-permeable membrane (filter) which collects large impurities, allowing the protein of interest to pass through. b) Diafiltration: This process is used to remove small molecules, such as exchange salts (used in chromatography), from the solution of interest. Solvent is typically added to the solution, which is then passed tangentially across a filter which collects/traps the small impurities as they go by the semi-permeable membrane, allowing the large protein molecule pass. This is done several times to achieve a desired purity. This type of filtration is also known as tangential flow filtration.
  20. 20. 5. Kinetics and thermodynamics of the process Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 17 5. Kinetics and thermodynamics of process 5.1. Kinetics The general aim in a biological reaction is to support the growth of a specific organism and to encourage a high product yield. It is therefore common practice to limit the concentration of essential nutrient to give controlled overall growth while provide others in excess. In this case, glucose is taken as the limiting reactant. All kinetic data were submitted to curve fitting techniques. An appropriate polynomial function was fitted by the least squares method to the measured concentration data. The derivative with respect to time was then calculated for the values obtained with the help of the polynomial function. The specific consumption or production rates could then be calculated by dividing the derivative by the viable cell concentration (Xv) at selected time points. The apparent specific growth rate, µ, was calculated using the following equation:
  21. 21. 5. Kinetics and thermodynamics of the process Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 18 The production of Rituximab belongs to category of fermentation wherein the production is due to recombinant technology and cell culture production of Chinese Hamster Ovary primary but the reaction rate is complex.. Hence proper conditions and control is required for maximum yield. Below the growth trend and substrate consumption for the CHO is given. Kinetics of CHO cell growth, Fig.1. Kinetics of CHO cell growth, in 96 well plates containing 200 mL of different media: serum-free reference medium (*), serum-free medium supplemented with 25 mg/L of sinapic acid (&), serum-free medium supplemented with 4 g/L of rapeseed peptide fraction (~) and serum-free medium supplemented with 25 mg/L of sinapic acid and of 4 g/L of rapeseed peptide fraction (_). Experiments were performed in triplicate
  22. 22. 5. Kinetics and thermodynamics of the process Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 19 5.2. Thermodynamics Biochemical pathways are complicated and it is not possible to find the ΔG values for every reaction taking place. But we can safely conclude that the overall reaction is thermodynamically feasible.
  23. 23. 6. Process Block Diagram Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 20 InoculumMedium Preparation Air sterilization Bioreactor Centrifugation Protein A Chromatography Ion Exchange Chromatography Cell mass Nutrient Air VIALS FILLING & PACKING Diafiltration FormulationStorage VR filtration Final Filtration
  24. 24. 7. Site Selection Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 21 7. Site Selection Proper site selection is one of the factors that govern the success of any project. The entire site selection exercise can be divided into three main factors:  Profitability factors: profitability of project  Prosperity factors: prosperity of nation  Productivity factor: productivity of plant 7.1. Profitability factors 7.1 .1.Availability of raw materials: Raw material contributes a major share to the operating cost of the project. This in turn is going to determine the cost of production of the product eventually deciding the profit margin. So it is important to consider this component in site selection. 7. 1.2. Market for the Product: The product Rituximab is used for cancer treatment, Hence the manufacturing site should be close to metro cities so that transportation cost is reduced 7.1.3. Land: Adequate land space must be available for all the buildings, units and equipments. Also provision for any future growth has to be considered. Other space requirements like effluent treatment plant or green belt also should be given due consideration. This land must be available at affordable price as this would eventually be contributing to the fixed cost of the project. 7.1.4. Soil Assay: This includes the type of soil, its bearing capacity, and identification of seismic zone and height of water table. Proper soil survey to avoid the mechanical breakdown of the plant is necessary. 7.1.5. Climatic conditions: The necessary climatic data like average rainfall, minimum and maximum temperature, frequency of cyclones and hurricanes should be considered while selecting a site. These have effect on the fixed cost of the plant.
  25. 25. 7. Site Selection Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 22 7.1.6 .Power: Regular and uninterrupted power supply at conceded rates is an essential factor to run the project smoothly. 7.1.7 .Water quality and availability: This is the most important parameter for site selection since water is required in huge quantities for the process. Water must be available in adequate amounts and with good quality at cheaper rates. Choosing a site which guarantees the uninterrupted supply of water having compatible qualities at affordable rate is desired. 7.1.8. Environmental considerations: Having a common effluent treatment plant in an industrial complex always helps reduce the load on the project. 7.2. Productivity factors 7.2.1 Communication: Communication facilities like Telephone, Fax, Telex, e-mails, etc. should be there at the site location. 7.2.2 Labour: Availability of skilled labour is important. 7.2.3 Infrastructure: The existence of well developed infrastructure is desirable. The site should easily accessible by road and railways. Also general civic amenities should be easily accessible.
  26. 26. 7. Site Selection Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 23 7.3 Prosperity factors 7.3.1 Appropriate Utilization of Raw materials: The natural resources of the country have to be utilized appropriately and efficiently. There should be proper utilization of resources, decreasing the load on the public transportation system. 7.3.2 Dispersal of Industries: Utilization of most of the available land, coupled with the objective of de-industrialization of metropolitan cities is an objective to be fulfilled by dispersal of industries away from residential zones which has lead to the development of this industrial estate. This calls for choosing a site which is located in specially secured industrial zones so as to take advantages of the facilities already present and keep distance from populated areas. 7.3.3 Security of the Nation: The site should not be situated in a politically sensitive area, so it does not endanger national security. 7.4.Site evaluation Based on the above mentioned factors the two sites which seem suitable to set up the manufacturing unit are Hosur, Krishnagiri district under TIDCO(Tamil Nadu Industrial Development corporation) (Site A) and Raigad under MIDC(Maharashtra Industrial Development corporation )(Site B).Both of these sites have been declared as biotech SEZ’s by the respective governments. Table 1. 1. Site Evaluation Criteria Site A Site B Land and site development 8 7 Building and civil construction 7 7 Climate 8 8 Raw Material source 8 10 Product Market 6 9
  27. 27. 7. Site Selection Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 24 Water/Effluent treatment 8 6 Power 6 8 Environmental Consideration 8 6 Housing and social community factor 7 8 Staff transport 5 8 Equipment transport 8 9 Taxes and subsidies 7 8 TOTAL 86 92 Therefore Raigad, Maharashtra is chosen as the manufacturing site.
  28. 28. 8. Material Balance Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 25 8. Material balance A] Inoculum preparation The inoculum is initially prepared in 225 ml T-flasks. The material is first moved to roller bottles (25 L), then to 50 L and subsequently to 75 L disposable bag bioreactors. Sterilized media is fed at the appropriate amount in all of these four initial steps (3.6, 11.4, 43.6, 175.4 kg/batch respectively). The broth is then moved to the first (25 L) and second (50 L) seed bioreactor. For the seed bioreactors the media powder is diluted using WFI in one prep tank. B] Bioreaction section Serum-free low-protein media powder is dissolved in WFI in a stainless steel tank (MP-103). The solution is sterilized using a 0.2 µm dead-end polishing filter (DE-103). A stirred-tank bioreactor (PBR1) is used to grow the cells, which produce the therapeutic monoclonal antibody (Rituximab). The production bioreactor operates under a fed batch mode. High media concentrations are inhibitory to the cells so half of the media is added at the start of the process and the rest is fed at a constant rate during fermentation. The concentration of media powder in the initial feed solution is 11.73 g/L. The fermentation time is 12 days. The volume of broth generated per bioreactor batch is approximately 50 L, which contains roughly 0.5 kg of product. Basis: Per batch Total working days = 300 days Total Product to be produced= 10 Kg (10mg/ml 10ml fill 100,000 vials) So 10 Kg to be produced in 300 days Total batches = 20 1 batch production = 0.5 kg Rituximab
  29. 29. 8. Material Balance Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 26 8.1. Bioreactor B001 Volume = 25 Liters Product Con = 11.73 g/l/day Total mass of cell = 11.73×V = 20×25=293.75g Total mass of cell in 1 batch ( 6 days) =1762.5g = 1.76 g The size of B001 is 34 Lit with 25 L working volume. Serum free media supplement 4.2 g/lit Total serum free media supplement = 4.2 × 25=105 g. Table 8. 1:Across B001 input(kg)(1) input(kg)(2) output(kg)(3) Cell mass 0.5 1 Serum free media 0 0.105 0 8.2. Bioreactor B002 Volume = 50 Liters Product Conc. = 11.73 g/l/day Total mass of cell = 11.73×V = 11.73×50=586.5g. Total mass of cell in 1 batch ( 6 days) =6000 mg = 6 g B001 1 3 2 2
  30. 30. 8. Material Balance Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 27 The size of B002 is 65 Lit with 50 L working volume. Serum free media supplement 4.2 g/lit Total serum free media supplemet =4.2 × 50=210 g. Table 8. 2: Across B002 input(kg)(3) input(kg)(4) output(kg)(5) Cell mass 1× 10-3 0 1.5 × 10-3 Serum free media 0 210 0 8.3. Material balance across the Production Bioreactor B003 The size of the production Bioreactor B001 is 0.065 m3 with 0.05 m3 working volume. Serum- free low-protein media powder is dissolved in WFI in a stainless steel tank. The solution is sterilized using a 0.2 µm dead-end polishing filter. A stirred-tank bioreactor is used to grow the cells, which produce the therapeutic monoclonal antibody (Rituximab). The production bioreactor operates under a fed batch mode. High media concentrations are inhibitory to the cells so half of the media is added at the start of the process and the rest is fed at a constant rate during fermentation. The concentration of media powder in the initial feed solution is 42 g/L. The fermentation time is 12 days. The volume of broth generated per bioreactor batch is approximately 80 L, which contains roughly 50 kg of product. B002 3 5 4 2
  31. 31. 8. Material Balance Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 28 Total cell mass=75 ×11.73=879.75g=879.75 g Amount of serum free media=4.2×75=315g. =0.315Kg. Amount of media required for composition = 0.8039 Time taken for it to get completely consumed Thus after every 7.14hrs, 0.8039kg of media needs to be supplied. SR.NO. COMPONENT mg/L(x) Conc (75*x) Kg 1 Amino Acid(20) 476.4 0.035 2 Vitamin (9) 3.6 270 × 10-6 3 Organic Compound(8) 1948.61 0.146 4 Inorganic Salts 8300.9 0.622 Total - 0.8039 Table 8.3: Across B003 input(kg)(5) Input(kg)(6) output(lit)(7) Amino Acid(20) 0.035 0 - Vitamin (9) 270 × 10-6 0 0 Organic Compound(8) 0.146 0 0 Inorganic Salts 0.622 0 0 Cell mass 0 879.75× 10-3 Serum free media 0 0.315 Total volume(lit) 0 0 72 B003 5 7 6 2
  32. 32. 8. Material Balance Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 29 8.3. Material balance across the Centrifuge C001 Between the downstream unit procedures there are 0.2 μm dead-end filters to ensure sterility. The generated biomass and other suspended compounds are removed using a Disc-Stack centrifuge. During this step, roughly 2% of Mab is lost in the solids waste stream resulting in a product yield of 98% for 3.8 hrs. Table 8.4: Across C001 input(7) output(8) output(9) % yield Mass of cell (g) 841.97 15.24 826.73 98 Volume(Lit) 71.78 1.3 70.48 98 8.4. Material balance across the Affinity Chromatography Column F001 The bulk of the contaminant proteins are removed using a Protein-A affinity chromatography column (C-101). The yield on Mab for this step is 90%.The protein solution is then concentrated 5x and diafiltered 2x (in P-21 / DF-101). The yield on product is 98% and this is represented by the product denaturation feature of the Diafiltration operation. The concentrated protein solution is then chemically treated for 1.5 h with Polysorbate 80 to inactivate viruses (in P-22 / V-111). C0017 9 8
  33. 33. 8. Material Balance Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 30 Table8.5: Across F001 input(9) output(10) output(11) % yield Mass of cell (g) 826.73 41.28 800.22 95 Volume(Lit) 70.48 3.52 68.22 95 8.5. Material balance across the Ion Exchange Chromatography Column F002 An Ion Exchange chromatography step follows (P-24 C-102) with a yield on Mab of 90%. Ammonium sulfate is then added to the IEX eluate (in P-25 V-109) to increase the ionic strength for the Hydrophobic Interaction Chromatography (P-26 C-103) that follows. 20% of Mab is lost during the HIC procedure. F001 9 11 10 F00211 13 12
  34. 34. 8. Material Balance Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 31 Table8.6: Across F002 input(11) output(12) output(13) % yield Mass of cell (g) 785.44 78.47 706.96 90 Volume(Lit) 66.96 6.69 60.27 90 8.6. Material balance across Virus Retentive Filtration F003S Table8.7: Across F003 input(13) output(14) output(15) % yield Mass of cell (g ) 706.96 70.61 636.35 90 Volume(Lit) 60.27 6.02 54.25 90 8.6. Material balance across Difiltration F004 F00313 15 14 F00415 17 16
  35. 35. 8. Material Balance Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 32 Table8.8: Across F004 input(15) output(16) output(17) % yield Mass of cell (g ) 636.35 31.78 604.56 95 Volume(Lit) 54.25 2.71 51.54 95 8.7. Material balance across Formulation Column FC001 Table 8. 9: Across FC001 input(gm) output(Kg)(20) Polysorbate-80(17) 37.97 0 Sodium Citrate dehydrate(18) 399.47 0 Sodium Chloride(19) 488.25 0 Cell mass(20)(g) 604.56 0.592 FC001 17 3 P00210 13 12 21 17 19 P00210 13 12 18 P00210 13 12 20 P00210 13 12
  36. 36. 8. Material Balance Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 33 8.8. Vials Filling Table8.10: Across V001 input Output Output % yield Mass of cell (g ) 592.36 5.86 586.5 99 Volume(Lit) 50.50 0.50 50 99 8.8. Summary of the entire process The material balance summary across all the units in the process is as follows Table8.11 : Material balance summary Purification Step %recovery at every step Centrifugation 98 Affinity Chromatography 95 Ion Exchange Chromatography 90 Virus retentive filtration 90 Dilfiltration 95 Formulation 99 Packing and storage 99 Total Process efficiency 69.64
  37. 37. 9. Energy balance Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 34 9. Energy balance 9.1. Across production Bioreactor B003 For sterilizing the Bioreactor and the Bioreactor media, it has to be heated to 121°C, be held at that temperature for a certain length of time and cooled to 37°C, the temperature at which fermentation takes place. Thereafter, fermentation being an exothermic process the temperature has to be maintained at 37°C by passage of cooling water through the coils 9.1.1. The heating cycle The vessel is heated from room temperature to 121°C by using saturated steam at 4bar pressure. The properties of steam at 4 bar are : temperature difference:121-30=91°C=365 k Cp: Specific heat capacity of Bioreactor media: 4.18 kJ/kg/°K m: mass of the substrate media:72 kg :Latent heat of condensation of steam at 4 bar: 2132.95 kJ/kg 9.1.2.The cooling cycle Cooling water at 28°C from the cooling tower is passed through the coils placed inside the vessel to cool it from 121°C to the Bioreactor temperature of 37°C. :Specific heat capacity of cooling water:4.18 kJ/kg : difference in cooling water inlet and outlet temperature:5°C :temperature difference of the Bioreactor media:121-37=84°C
  38. 38. 9. Energy balance Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 35 9.1.3.Maintaining of Bioreactor temperature at 37°C Bioreactor is an exothermic process. The metabolic heat generation rate is assumed to be 10 kW/m3 (Biotranformation and bioprocess,pg 182).The heat removal rate should be 115 kW. Therefore, flow rate of cooling water required is 5.5 kg/s 9.5. Across fired heater E001 Air at room temperature of 30°C and 40 % relative humidity is heated by passing through the tubes of coal fired heater to 110°C CV: Calorific value of coal :20,000kJ/kg.
  39. 39. 10. Equipment Design Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 36 10. Equipment design 10.1. Process Design of Bioreactor 10.1.1. Mode of operation As can be seen in Section of material balance intermittent supplies of media is required to maintain the cell growth composition. Since it is aerobic fermentation, continuous supply of oxygen is essential. Considering the difficulty of controlling and maintaining monosepsis condition in a continuous mode, fed batch mode is used. 10.1.2. Bioreactor type The type of Bioreactor depends on the nature of the process (including cell kinetics), operating conditions (namely, mode of operation and gas liquid flow patterns), and physical and chemical properties of the substrates and the microbe. For the required production capacity, volume of substrate media necessary in the bioreactor is 0.075 m3 .For this scale of operation a mechanically stirred tank reactor consumes less power per unit volume as compared to non-mechanically agitated system. 10.1.3. Bioreactor geometry (Walas,1990, Pg no .288) Assuming liquid medium occupies 75% volume Volume of the Bioreactor: The height to diameter ratio for the vessel is taken as 2 because  The system has low viscosity(<25 Pa s)  The volume required for fermentation is not very huge  The system is shear sensitive. Hence less number of impellers should be used  
  40. 40. 10. Equipment Design Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 37 10.1.4. Impellor type and design The functions to be performed by the impeller in the system are  Mixing  Effective dispersion of air The controlling parameter is the oxygen dispersion in there for which radial impellers are preferable. However they consume high power. Hence we use a mixed flow pitched blade turbine impeller. Let the d:D be 1:13 and W:D be 1:5 For the CHO cell under the process conditions of 72 L substrate volume (Dtank=215mm) and 500 rpm impeller (3 six blade disc turbine dimp=80mm) speed, the air flow rate for optimal production is found to be 1 vvm (El Enshasy ,H. et al,2008) ( ) During scale up ,the objective is to maintain the same mass transfer coefficient of oxygen. For pilot scale At this calculated , is 1.37 ( ) 6.515W s
  41. 41. 10. Equipment Design Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 38 For production scale ( ) s 10.1.5.Sparger design Gas hold up in the system (Handbook of chemical engineering calculations, Pg no. 575) ( ) = volumetric flow rate of air = 0.2 m3 /s Notations: D: Diameter of tank d: Diamter of impeller W: Width of impeller : density of the medium : Volumetric air flow rate N : speed of impeller : gas hold up in the system V :volume of the Bioreactor.
  42. 42. 10. Equipment Design Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 39 10.2.Protein A CHROM COLUMN Protein A is a cell wall component produced by several strains of Staphylococcus aureus. The 46.7kDa-protein consists of a single polypeptide chain that is essentially devoid of carbohydrate. Native Protein A has contains four high-affinity (Ka = 108 /mol) binding sites that are capable of interacting with the Fc region of IgG-class antibodies from selected mammalian species. IgG- binding function is optimal at pH 8.2, but efficient binding also occurs in neutral and physiological buffers (pH 7.0 to 7.6).  Native Protein A – immobilized Protein A is ideal for polyclonal IgG purification.  Agarose resin – support is crosslinked 6% beaded agarose (CL-6B), the most popular resin for protein affinity purification methods. Properties of crosslinked 6% beaded agarose (CL-6B):  Support pH Stability: 2 to 14 (short term); 3 to 13 (long term)  Average Particle Size: 45 to 165 microns  Exclusion Limit: 10,000 to 4,000,000 daltons  Maximum Volumetric Flow Rate: approx. 1mL/minute.  Maximum Linear Velocity: 30cm per hour  Maximum Pressure: less than 1.5 bar, defined as the maximum pressure drop across a column that the resin can withstand (Note: The indicated gauge pressure of a liquid chromatography apparatus may be measuring the total system pressure rather than the pressure drop across the column.)  Inert and stable – superior manufacturing method immobilizes Protein A by charge-free, leach-resistant covalent bonds, resulting in low nonspecific binding and enabling multiple uses without decline in yield  High capacity – this “Plus” variety of Pierce Protein A Agarose has a dense load of immobilized Protein A, providing a binding capacity greater than 34mg human IgG/mL resin (approx. 16 to 17mg mouse IgG/mL resin)
  43. 43. 10. Equipment Design Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 40  Multiple formats – choose from bottled resin slurries, centrifuge-ready columns, complete purification kits, two sizes of FPLC-ready chromatography cartridges, and 96- well filter plates. 10.3. Process Design of Diafiltration Unit Is a technique that uses basic principles of filtration to completely remove, replace or lower the concentration of salts or solvents from solutions containing biomolecules, Uses permeable membrane to separate the components mainly based on size and column-based gel filtration.
  44. 44. 10. Equipment Design Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 41 10.3.1. Design Consideration a) Type of flow (tangential vs. direct) Direct Flow: - Large molecule trapped on membrane and forms gel - More susceptible to fouling - Flux rate decreases as volume filtered increases
  45. 45. 10. Equipment Design Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 42 Tangential Flow - Solute diffuses through the surface of the membrane tangent to the flow of the feed - Minimize buildup of molecules – less fouling - Prevents rapid decline in flux rate. Reference:-Millipore Inc., 2003 b) Membrane selection Primarily based on size of biomolecule and Molecular weight cut off (MWCO) of the membrane should be 1/3rd to 1/5th of the MW of the molecule to be retained,Typical MW of mAb: 150kDa => 30000 MWCOO,For protein separation: 30 LMH. c) Type of diafilter modules i) Flat sheet tangential flow ii) Hollow fibre iii) Tubular iv) Spiral wound
  46. 46. 10. Equipment Design Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 43 d) Continuous vs. discontinuous flow Continuous  Typically constant volume  Removal rate of salt = addition rate of water  Addition of WFI is at 1/3rd of the removal rate of salt (filtrate).  More suited for process scale- requires pumps Discontinuous  Concentration and dilution cycles  Usually more feasible on a laboratory scale
  47. 47. 10. Equipment Design Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 44 9.2.2. Final design Feed flow rate = 3L/min/m2 -Hollow fiber cartridges Membrane area needed Mem. area = filtrate vol / (filtrate flux * process time) = 50/(30x2)=0.83 m2 Pump Feed rate (L/min) = Feed flow rate X Area = 3 X 0.83=2.49 L/min -UNIT NUMBER: Stainless stain housing 88 90 92 94 96 98 100 102 15 17 19 21 23 25 27 29 0 1 2 3 4 5 6 PercentofAceticAcidremoval MembraneArea(m2) Diafiltration Volume Effect of diafiltration volume on membrane area requirement and percent of acetic acid removal Membrane Size Removal of Acetic Acid
  48. 48. 10. Equipment Design Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 45 Steam in place cartridges can be added Reference :- GE Healthcare (2007)
  49. 49. 11. Mechanical design Of Bioreactor Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 46 11. Mechanical Design of Bioreactor B003: H =0.41m D= 0.39m 11.1. Thickness of vessel required Max pressure P = 1.013 bar during cell growth.. Hence design pressure =1.013 *1.4= 1.418 bar = 0.141 N /mm2 t = 0.141× 390 / ( 2× 140 × 0.85)-0.141) + 2 = 2.23 mm We take thickness 6mm as this must be same as head thickness. 11.2. Design of Heads: At the top: A torispherical head is used . It is connected to the shell by means of a flanged joint. Crown Radius Rc = 390 mm Knuckle Radius R1 = 24 mm (6% of Rc) The thickness of the torispherical head is given as 2.f.J .WP.R t c h  + C where W = stress intensification factor ] R R 3[ 4 1 1 c  = 1.77
  50. 50. 11. Mechanical design Of Bioreactor Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 47 J = joint efficiency = 0.85 ( flange joint)  th = 0.41+2 = 2.41 mm At the bottom: A torispherical head is welded to the shell at the bottom end. Here J = 1 Applying the same formula , thickness = 3.12 mm , So we use 4 mm thickness 11.3. Design of Flanges for Head and Shell Internal gasket diameter, Gi = 390 +10 = 400mm External gasket diameter is calculated as; pmpY mpY G G a a i o    Gasket seating stress, Ya =52.5 N/mm2 Gasket factor (m) =3.75 0013.1 i o G G Go = 400.54mm A flat asbestos gasket of 390 mm internal diameter and 400 mm external diameter is used. Gasket seating width = b = (400-390) ×0.5 = 5 Under atmospheric conditions bolt load due to gasket reaction is given by:
  51. 51. 11. Mechanical design Of Bioreactor Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 48 Wm1 =  b G Ya Where G = ( Gi + Go)/2 = 800.54 mm Gasket seating stress, Ya =52.5 N/mm2  Wm1 = 6.6 x 105 N The load under operating conditions is given by Wm2 = .P.G 4 b).G.m.P2.( 2   = 8.42x 104 N The total bolt area is calculated on the basis of the greater load  A = f Wm1 where f is the permissible stress in bolt (138 N/mm2 ) Therefore, A = 41,826 mm2 Let us use bolts of area =500 mm2 Hence number of bolts required =78. Bolt area = 78*d 4 2 Hence bolt diameter = 28.56 mm So we use M26 bolts ( 78 Nos ) Pitch circle diameter (P.C.D) = Outside diameter of gasket + 2  diameter of bolt + 12mm = 400+ 2*28.56 +12 = 469.12 mm
  52. 52. 11. Mechanical design Of Bioreactor Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 49 Flange thickness is given by fK P Gtf  where HG hW5.1 3.0 1 K gm   Wm = total bolt load = 6.6 x 105 N hg = Radial distance from gasket load reaction to basket circle = (PCD - G)/2 = 34.5 mm H = Total hydrostatic end force = PG 4 2 = 0.6 x 106 N K = 2.69 tf = 44.6 mm Use 50 mm thickness of flange. 11.4. Design of Nozzles: The nozzles provided are as follows: 1) On shallow dished head at top:  200 mm nozzle for substrate inlet.  50 mm nozzle for pressure indicator  50 mm nozzle for pH indicator
  53. 53. 11. Mechanical design Of Bioreactor Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 50  50 mm nozzle for thermocouple  50 mm nozzle for acid inlet  50 mm nozzle for antifoam  50 mm nozzle for Pressure relief valve  5 mm nozzle for level detector  450 mm nozzle for manhole.  150 mm nozzle for sight glass. 2) On torispherical head, at the bottom:  20 mm nozzle for draining out broth  10 mm nozzle for Air inlet 3) On shell:  30 mm nozzle for coil inlet  30 mm nozzle for coil outlet For 30 mm nozzle on shell: Nozzle thickness required, tn = P-2fJ dP i =0.154*300/(2*1*140 - 0.154) = 0.165 mm Actual thickness taken =6 mm.The area for which compensation is required is A = d ts` = 300 x 1.42 = 426 mm2
  54. 54. 11. Mechanical design Of Bioreactor Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 51 Area available for compensation: i) Area of compensation provided by the portion of the shell as excess thickness Ah = d (ts – ts`) Where ts` = theoretical shell thickness required = 1.42 mm ts= Actual shell thickness used = 6mm d = Diameter of nozzle As = 1374 mm2 ii) Area of compensation provided by the nozzle: An = 2 x Ho x (tn - tn`) tn = Actual thickness of nozzle used = 6 mm tn` = Nozzle thickness required theoretically =0.165 mm Ho = 2.5 x tn An = 29.175 mm2 As + An =1181.175 mm2 > A So reinforcement ring is need not be provided.From similar calculations, it is found that no reinforcement ring is required for any of the nozzles. 11.5. Design of supports Selection of support: Vertical vessels : Bracket or Skirt support H/D: 2 to 5 - Bracket support
  55. 55. 11. Mechanical design Of Bioreactor Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 52 H/D>5 – Skirt support Hence, we use Bracket support No. of brackets for D< 3000mm = 4. i) Thickness of stiffener ( horizontal plate) Wmax = Σ W / no. of brackets (neglecting wind loads as vessel is indoors and is not very tall) = (11.5*1200)*9.8 /4 =33,810 N               44 4 max * 7.0 Lb b t L lb W f h h Where, fh = permissible bending stress = 155 N/mm2 l= 75 mm b=h=200 mm L= 300 mm Hence , th = 0.51mm th = 2 mm ii) Thickness of Gusset plate   COShf lW t h g 2 max3
  56. 56. 11. Mechanical design Of Bioreactor Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 53 Θ = 45 o Hence tg = 1.22 mm We take tg = 2 mm 11.6. Design of shaft : Diameter of agitator = 700 mm Agitator Speed = 151 rpm Power = 3.6 kWatt The continuous average torque on the shaft is given by, Tc= power / (2xxN), = 229.18 N-m The shaft must be capable of resisting 1 ½ times the continuous average torque Tm = 1.5 x Tc = 344 N-m = 34400 N –cm Zp = s m f T fs = Maximum permissible shear stress on shaft ( 9457 N / cm2 ) Zp = Polar modulus of section of the shaft Zp = 16 3 d (×d3 )/16= 3.63cm3 d = diameter of shaft
  57. 57. 11. Mechanical design Of Bioreactor Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 54  d = 2.65cm Hence, we use a shaft of 6 cm diameter.
  58. 58. 12. Equipment Sizing Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 55 12. Equipment sizing 12.1. Bioreactor B001 To ensure 5% by volume inoculum at every stage of fermentation, the size of B001 is taken as 0.025 m3 . D:Diameter of tank h:Height of tank Table 11.1 :Sizing of B001 Type Jacketed stirred tank vessel Operating temperature 370 C Operating pressure 1 atm Volume 0.025m3 Diameter 0.3m Height 0.3m MOC SS316 12.2.Seed Bioreactor B002 The sizing is done as described in Section 11.1 Table 12.2:Sizing of B002 Type Jacketed stirred tank vessel Operating temperature 370 C Operating pressure 1 atm Volume 0.05m3 Diameter 0.4 m Height 0.4 m MOC SS316
  59. 59. 12. Equipment Sizing Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 56 12.3.Production Bioreactor B003 The sizing is done as described in section 11.1 Table 12.3 : Sizing of B003 Type Jacketed stirred tank vessel Operating temperature 370 C Operating pressure 1atm Volume 0.075 m3 Diameter 0.5m Height 0.5m MOC SS316 12.4.Centrifuge C001 The quantity of supernatant to be processed is 0.07 m3 . Let the flowrate through the centrifuge be 0.25m3 /hr(Perry’s).Time taken for complete centrifugation= . Settling velocity in presence of centrifugal force according to stokes law :settling velocity of particle under gravity (m/s): m/s G: ratio of settling velocity under centrifugal force to that under gravity: 4300(assumption). :diameter of cell particle(m):2µm :density difference between cell particle and water:600kg/m3 :Vicosity of supernatant(Pas):8.5× Pa s Area required for centrifugation=
  60. 60. 12. Equipment Sizing Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 57 Table 12. 4. Sizing of C001 Type Disc stack centrifuge Operating temperature 30º C Operating pressure 1 atm G 4300 Area 53.41 m2 Diameter 0.5m No.of discs 12 MOC SS316 12.5. Protein A CHROM COLUMN F001 Type Protein A CHROM COLUMN Column Agarose resin, 4.6mm ID × 10cm Eluent A] 20mmol/L MES buffer, pH 6.0 B]0.5mol/L NaCl in 20mmol/L MES buffer, pH 6.0 Gradient 0min (10%B), 15min (30%B), linear Flow rate 1.0mL/min Temperature 250 C Injection volume 20μL Samples A: human antibody, IgG1 B: human antibody, IgG1 Sample concentration 0.5g/L
  61. 61. 12. Equipment Sizing Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 58 12.6. ION EXCHANGE COLUMN F002 12.6.1 Anion Exchange column STREAMLINE Q XL, 7.5 l Certificate of Analysis Yes Flow Velocity <500 cm/h Matrix 6% cross-linked agarose containing a quartz core with dextran surface extender Storage Conditions 4 to 30°C, 20% Ethanol Chemical Stability All commonly used buffers, nonionic detergents, 1 M NaOH, 6 M guanidine hydrochloride Ligand Quaternary amine Ion Exchanger Type Strong anion exchanger Ionic Capacity 0.23-0.33 mmol Cl- /ml medium pH Stability Cleaning- in-Place (CIP) 2-14 BioProcess Medium Yes Average Particle Size 200 µm Binding Capacity/ml Chromatography Medium > 110 mg BSA/m medium pH Stability Working Range 2-12 Particle Size 100 µm-300 µm
  62. 62. 12. Equipment Sizing Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 59 12.6.2. Cation Exchange column STREAMLINE SP XL Chemical Stability All commonly used buffers, nonionic detergents, 1 M NaOH, 6 M guanidine hydrochloride Flow Velocity <500 cm/h Storage Conditions 4 to 30°C, 20% Ethanol + 0.2 M Sodium Acetate Ionic Capacity 0.18-0.24 mmol H+ /ml medium Average Particle Size 200 µm Matrix 6% cross-linked agarose containing a quartz core with dextran surface extender Ion Exchanger Type Strong cation exchanger Particle Size 100 µm-300 µm Binding Capacity/ml Chromatography Medium > 140 mg lysozyme/ml medium Ligand Sulphopropyl pH Stability Cleaning- in-Place (CIP) 3-14 pH Stability Working Range 4-13 Certificate of Analysis Yes BioProcess Medium Yes
  63. 63. 12. Equipment Sizing Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 60 12.7.Virus retentive filtration F003 The product concentration in the system =11.73 g/L.It is concentrated 5 times.An optimum flux of 0.000025m/s is obtained at a trans membrane pressure of 72.4kPa.Let process take 4 hrs. Table 12.5 .Sizing of Filtration F003 Type Tubular membrane unit Operating temperature 30º C Transmembrane pressure 72.4 kPa Area 1.64m2 No of modules 16 MOC Regenerated cellulose membrane , SS304 cover 12.8. Diafiltration Unit F004 The protein concentration in the system 2.5 wt %(24.62 kg/m3 ).It is concentrated 5 times. An optimum flux of 0.00025 m/s is obtained at a trans membrane pressure of 72.4kPa. Table12.6.Sizing of Filtration P004 Type Tubular membrane unit Operating temperature 30º C Trans membrane pressure 72.4 kPa Area 6.56m2 No of modules 58 MOC Polysulfone membrane , SS304 cover
  64. 64. 12. Equipment Sizing Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 61 12.9. Air Compressor S0001 Compressed air is required for fermentation. Air flowrate:70.92m3 /hr Air inlet pressure:1.5 bar Table 12.7. Sizing of S0001 Type Single stage axial flow compressor Power 4HP Capacity 0.02 m3 /s Suction pressure 1 bar Discharge pressure 2bar MOC SS304 12.10.Pumps. Different pumps are used at various stages in the process. Each pump is attached with a storage vessel for liquid. They are as follows Table 12.8. Sizing of pumps Function Capacity(m3 /hr) Discharge pressure(bar) Power(HP) Type P001 2.6 1 0.096 Centrifugal P002 3.25 2 0.24 Centrifugal P003 3.09 1 0.115 Centrifugal P004 0.138 1.724 0.00885 Centrifugal P005 0.025 1.724 0.0016 Centrifugal P007 0.00483 1.724 0. Centrifugal
  65. 65. 13. Instrumentation and Process control Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 62 13. Instrumentation and process control In any process, instrumentation and control plays an important role to help achieve the desired degree of productivity. In a bio-fermentation process, it is important to maintain certain parameters at a specific value for the optimum growth of cells. The basis control strategy involves a sensor which measures the process variable and converts it into appropriate signal via the transmitter .The transmitter then sends the signal to an indicator and controller to which set point is fed for the controlled variable. The indicator and controller compare the values and depending on its type execute a controlling action through the final control element which is often a pneumatically or electrically controlled valve. The control hardware is either analog or digital. The analog system may be pneumatically operated using instrument air or electrically through wire. 13.1. Different types of controllers 13.1.1 P controller The proportional controller actuates the output proportional to the error. It gives considerable offset but is simple and cheap and fast. 13.1.2. PI controller It is a proportional plus reset controller. The integral action causes the controller output to change as long as there is error in the output. Such a controller can eliminate small errors. It produces zero offset but response in oscillating and sluggish 13.1.3. PID controller It is a proportional plus reset plus rate controller. It anticipates what the error will be in future and applies a control action proportional to the rate of change of error. However for response with constant non-zero error it gives no control action. Also for small errors it may unnecessarily bring about large control actions.
  66. 66. 13. Instrumentation and Process control Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 63 13.2. Control strategy for the Bioreactor 13.2.1. Temperature Control Fermentation being an exothermic process, jacketed cooling water flow is used to maintain the temperature of the fermentor at 37°C. This is done using a cascade control loop with the aid of TIC01(primary controller) and FIC02(secondary controller ).PID controller is used since no offset can be tolerated. Measured variable: Temperature of Bioreactor, flow rate of cooling water in the jacket Controlled variable: Temperature of Bioreactor Manipulated variable: Flow rate of cooling water 13.2.2. Pressure Control The control of pressure of the system is important from the safety point of view and to maintain sterile conditions. During the fermentation process an internal pressure of about 1.2 bars is desired to maintain sterile conditions. Although the pressure may go up to 1.3-1.4 bar during the sterilization. Here a split range control loop is used controlling both the inlet air flow and the vent flow rate through PIC01 to maintain a stipulated pressure inside the fermentor.PID controller is used. Measured variable: Pressure inside Bioreactor Controlled variable: Pressure inside Bioreactor Manipulated variable: Vent air flow rate, Inlet air flow rate 13.2.3. pH Control The pH of the system needs to be maintained at about 6-7. As the fermentation proceeds the pH of the broth continues to increase. The pH is maintained by the periodic addition of sulfuric acid using a feed-back control strategy wherein the pH indicator pHI01 is connected to a controller FIC01controlling the flow of acid into the system. Here a PI controller is used.
  67. 67. 13. Instrumentation and Process control Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 64 Measured variable: pH inside Bioreactor Controlled variable: pH inside Bioreactor Manipulated variable: Acid Flow rate 13.2.4. Control of foam formation There may be a rise in the liquid level due to foam formation. This is controlled by the addition of anti-foam when the liquid level (foam level) reaches a predetermined level. When the foam reaches the probe tip, a current is passed through the circuit of the probe which gets completed, with the foam acting as the electrolyte and the vessel as earth. Here again a feedback control strategy is used wherein signals of level indicator are used to control the antifoamer flow rate. A timer is also provided to ensure enough time for the antifoam to mix properly before more anti- foam is added. A PI controller is used. Measured variable: level inside Bioreactor Controlled variable: level inside Bioreactor Manipulated variable: Flow rate of antifoamer.
  68. 68. 13. Instrumentation and Process control Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 65 TT: Temperature transmitter FT: Flow transmitter LT: Level transmitter PT: Pressure transmitter PIC: Pressure indicator and controller TIC: Temperature indicator and controller
  69. 69. 14. HAZOP Studies Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 66 14. Hazard and Operability (HAZOP) Studies The HAZOP study is a formal procedure to identify hazards and operational difficulties for a given process and be prepared with corrective action for the same. The various terminologies used in HAZOP analysis are: Parameter: The important controlling factor. Guidewords: The different guidewords used and their significance is as follows: Table14.1. Guide words Guide Words Meaning Comments None The complete negation of the intention No part of the design intention is achieved. High, Too High Quantitative increase Applies to quantities such as flow rate and to activities such as heating Low, Too Low Quantitative decrease Applies to quantities such as flow rate and to activities such as heating Possible causes: Probable reasons for increase/decrease of parameter. Possible consequences: Probable repercussions of increase/decrease of parameter. Actions: Immediate activity to be performed to bring back normal functioning. Safeguards: What should be done to prevent operating problems in the future .
  70. 70. 14. HAZOP Studies Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 67 The table shown below presents HAZOP study done for the Bioreactor (B003): Table 14. 2. HAZOP of B003 Parameter Guidewords Possible causes Possible consequences Actions Safeguards Cooling water flow rate Too high 1. Failure of thermocouple. 2. Failure of control valve. 3. Failure of controller. Decrease in temperature - Not optimum for microbes 1. Manually decrease flow rate 2. Increase cooling water temperature 1. Install extra valve (Bypass) 2. Install alarm Too low 1. Failure of thermocouple. 2. Failure of control valve. 3. Failure of controller. Increase in temperature - poor Yield 1. Manually increase flow rate 2. Reduce cooling water temperature 1. Install extra valve (Bypass) 2. Install alarm Cooling water temperature Too high Failure of refrigeration system Increase in temperature- poor Yield Increase flow rate 1. Install cascade control 2. Install Alarms Too low Setting error in refrigeration system Decrease in temperature-Not optimum for microbes Decrease flow rate 1. Install cascade control 2. Install Alarms
  71. 71. 14. HAZOP Studies Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 68 Impeller Speed Too high Operator or mechanical error High Shear- destruction of microbes. Manually reduce impeller speed Install alarm Too low Operator or mechanical error Poor distribution of air. Manually increase impeller speed Install alarm None Power failure or failure of impeller No distribution of air and no suspension of microbes. Switch on backup power supply. 1. Install generator 2. Install alarm Internal pressure Too high 1.Failure of pressure valve 2. Failure of pressure controller Explosion 1. Open pressure relief valve manually 2. Shut-off air flow. 1. Install a critical alarm 2. Install disc valve that automaticall y releases pressure. Too Low Vent open / leakage in valve Contamination Seal valve. 1. Install an extra valve 2. Regularly carry out maintenance of valve.
  72. 72. 15.Batch Scheduling Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 69 15.Batch Scheduling Proper batch scheduling is important for achieving optimal productivity.Bioreactor is carried out in fed- batch mode. Using the unsteady state equations,the time required for various activities is found as follows For sterilization of Bioreactor B002 Heat up time =36 min. Hold time = 45 min. Cool down time =10 min . Table 15.1. Batch Scheduling Activity Time(hrs) Initial Down time for Transformation/ Preparation 5 B001 Fermentation Down time TOTAL 48 2 50 Activity Time(hrs) BOO2 (50L) Fermentation Down time TOTAL 60 5.75(considered for lag phase, preparation etc) 65.755 B003 Heating Hold time 0.6 0.75
  73. 73. 15.Batch Scheduling Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 70 Cooling Fermentation Emptying TOTAL 0.1666 143.69 1.45 146.66 Centrifuge C001 1 F001 Affinity Chromatography 0.5 F002 Ion exchange Chromatography 0.5 F003 VRF 4 F004 UF 4 F005 DF 4 FC001 Formulation column 1 The total time taken for the first batch is 282.41hrs.(11.76 days).Thereafter all the batches take 282.41hrs
  74. 74. 16.MOC Selection Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 71 16. MOC Selection For efficient working of the process, the stability of equipment is very important. This stability to a large extent depends on its material of construction. The bioreactor vessel is made of steel, metal or their combination. The relation between the height H and diameter D of the bioreactor is within 1.5-2.5, There are high requirements for the reactor vessel materials to prevent the inhibition of the microorganism growth. The same applies also to any other part (sensors, pipes, etc.), which are installed inside the bioreactor vessel. Few important characteristics to be considered when selecting a material of construction are: 16.1. Mechanical properties This includes parameters like tensile strength, stiffness elastic modulus (Young’s modulus), toughness , hardness , fatigue resistance, creep resistance. The selected MOC needs to have good mechanical properties under the process conditions. During sterilization all the equipments are exposed to intense temperatures and pressure. Hence the material chosen should possess superior mechanical properties. 16. 2. Corrosion resistance This is an important characteristic to be considered while choosing an MOC for the considered process. The presence of aeration coupled with agitation and tendency of reduction in pH during the process, demand a material with high corrosion resistance. To improve corrosion resistance the chromium content in the material needs to be above 12 %. 16.3. Contamination. For any biological process, highly aseptic environment is required. In industries such as the food, pharmaceutical, biochemical, and textile industries, the surface finish of the material is as important as the choice of material, to avoid contamination. 16.4. Cost The material cost plays an important role in project cost evaluation. Hence an optimal choice should be made which justifies the trade off between the cost and quality of material chosen.
  75. 75. 16.MOC Selection Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 72 Based on the above criteria, All the metal parts should be made from stainless steel. The most widespread brand of the stainless steel applied in bioreactors is 316L. The letter L indicates that this steel is with a low composition of carbon. The inner surface of the stainless steel bioreactor should be polished to about a mirror surface quality to facilitate the washing and sterilization process. Welding should be carried out in a fully inert gas medium. The inert gas should be argon, which fully replaces the air. With time, the application of the welding technology not corresponding to the requirements can cause the corrosion of the welds. The MOC selected for all other equipments in the plant is stainless steel.
  76. 76. 17. Plant Layout Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 73 17. Plant Layout: The general manufacturing site layout is as shown below: Fire Station Time Keeping Office Securit y Office Green Belt Administration Building Canteen Rest Room Securit y Office Records Keeping Workshop And Maintenance Storage Tanks Medical Facilities Control Room Store Room MAIN PLANT AREA Reserved Space For future growth 10 Water Chilling Plant Parking Green Belt ETP ETP Steam Generation
  77. 77. 17. Plant Layout Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 74 The equipment layout in the plant is as follows: B003 S002 S004S003S001 B001 B002 S0001 S005 C001 F001F002F003F004FC001 V001 PRODUCT STORAGE S006
  78. 78. 18.Financial Analysis Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 75 18. Financial Analysis 18.1. Project cost estimation. For estimating the Project Cost (Fixed Capital Investment), we first estimate the Cost of equipment (Delivered), The table below displays the key economic evaluation figures for the case of Rituximab. 18.1.1. Operating cost Breakdown The sizing of all the equipments is shown in Chapter 11 Equipment Design and MOC selection is discussed in chapter 12 Costs of equipments (plant and machinery) can be deduced as a direct function of the size of that equipment and its material of construction, which are obtained from formulae, cost – capacity correlations, graphs, etc. In cases where all data pertaining to the equipment isn’t available, the following power – law model is used to deduce the cost of the equipment. n= 0.68: For general equipment. 0.63: For Fluid handling equipment. 0.63: For vessels, storage units etc. Also, to take into consideration the time face the CE plant cost index needs to be taken. Table 18.01Costs of various materials Sr. Material Cost (Rs./kg) No. (including fabrication) 1. Carbon Steel 150 3. SS-316L 550
  79. 79. 18.Financial Analysis Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 76 18.1.11 Gross Cost of installed equipment Table 18.11: Gross cost of installment Equipment Installmnent Cost (Rs) 2.2 lit Roller Bottle 1800 DFT DEF cartridege 300000 225 ml T flsk 1200 DFT membrane 48000 100 lit cell bag 1260 Viral exclusion membrane 1602720 20 lit cel bag 24000 Protin A column 6480000 Ion exchange column 7200000 B001 1000000 B002 1800000 B003 1500000 C001 300000 F001 400000 F002 400000 F003 400000 F004 1200000 FC001 500000 V001 1000000 Freeze 1200000 Storage tank 500000 Compressor 1500000 Pump
  80. 80. 18.Financial Analysis Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 77 The gross cost of installed equipment = Rs. 273.5898 Lakhs. 18.2 Estimation of plant and machinery cost 18.2.1 Equipment cost for Outside Battery Limit (OSBL) facilities The OSBL facilities required are:  Cooling unit for Dynalene MS 2    Storage facility for raw materials and products    Cooling Tower  The Battery Limit plant has few pieces of equipment. Therefore, it would be reasonable to assume that the cost of the OSBL facilities would be a fraction of the Battery Limit plant. We assume that it is 30 % the cost of battery-limit equipment. In a similar way the piping, instrumentation, electrical, installation and other components of plant and machinery costs are estimated. 18.2.2 Plant and Machinery cost Table18.12: Breakup of OSBL Component % of Equipment cost Cost (lakhs) Equipment 273.5898 Piping 20 54.71796 Instrumentation 20 54.71796 Electricity 15 41.03847 OSBL 30 82.07694 Total A 506.1411 Spare 5 % 0f A 25.30706 Subtotal B 531.4482 Packaging and forwarding 3 8.207694 Transportation 3 8.207694 Insurance 1 2.735898 Installation 15 41.03847 insulation and painting 2 5.471796 VAT 10 27.35898 Octrai 3 8.207694 Excise 15 41.03847 GRAND TOTAL plant and machine( Lakh) 673.7149
  81. 81. 18.Financial Analysis Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 78 18.2.3 Estimation of total project cost The project can be estimated based on rule of thumb by breaking up the project cost components as the percentage of overall project cost. (Mahajani & Mokashi, 2005). Table 18.13 Breakup of Total Capital Cost Component % Cost Land and site development 10 134.743 Building and civil work 8 107.794 Plant and machinary cost 50 673.715 Khow how and engineering fees 8 107.794 Miscellaneous Fixed Assets (MFA) 3 40.4229 Contingency 5 67.3715 pre operative expenses 13 175.166 Preliminary & Capital issue related Expenses 3 40.4229 Total Project Cost (Lac) 100 1347.43 Total Project Cost = Rs 1347.43Lakh = Rs 13.47Crores Note: Price of Land is found from G.I.D.C website Land and site development cost 134.7429765 Lakh GIDC Rate 1500 Rs/m2 Area of land 8982.8651 m2 Area of land 2.201682623 Acres
  82. 82. 18.Financial Analysis Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 79 20. Estimation of Working Capital Working capital is the capital required to make the project work perform. The estimates are based on the guideline by Mahajani & Mokashi (2005). The following are the components of working capital. 20.1 Raw Materials We generally keep an inventory of 1 batch as storage of Raw Materials as work in progress. Steam is also generated in plant from waste heat boiler and air is compressed whenever required so no need to store it. The inventory, therefore, is not applicable for raw materials. 20.2 Product and Utility in stock The amount is estimated at the cost of production. Table 20.1 Product Streams as WIP and Utility costs for 1 batch Product No. of days Amount(kg) Cost(Rs)/Kg Total Cost(Rs) Product 15 0.5 12000000 6000000 0 Utilities 15 5 % of total 315789.4737 TOTAL PRODUCT COST ( Rs) 6315789.474 20.3 Maintenance Spares Calculating the plant and machinery item of project cost for 1 month. The total plant and machinery cost of the project = 673.7149 lakhs. A provision is made for maintenance spares as 0.1% of the plant and machinery item of the project cost for one month. Thus, maintenance spare inventory cost = Rs. 0.67374883 lakhs. 20.4 Other fixed costs These are estimated as 10% of the product inventory cost. Thus, other fixed expenses = 10% of (644) lakhs. = Rs. 6.31578947 lakhs.
  83. 83. 18.Financial Analysis Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 80 Table 20.2 Total working capital break-up Parameter Cost(lakh) Raw material inventory cost 49.232858 Product inventory cost 63.1578947 Thus, maintenance spare inventory cost 0.67371488 Other fixed cost 6.31578947 TWC (Lac) 119.380257 20.5 Working capital source Following table shows source of total working capital. Table 20.3 Source of working capital Component Contribution Cost in lakh Borrowed WC 75 % TWC 89.53519 Margin money 25% TWC 29.84506427 Total Working Capital ( Lac) 119.3802571
  84. 84. 19. Total Cost of Production Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 79 19. Total Cost of Production After finding the Total Capital Investment, we need to estimate the cost of production of each kg of the product. This has significant impact on selling price and hence the profitability of the project. Following are the main components of cost of production. It is assumed that the plant runs continuously for 300 days in a year. 19.1 Raw Material Cost Table 19.1: Cost of Raw Materials Raw material Batch req (kg) Rate(Rs/kg) Annual req(kg) Cost (Rs) Inoc. Media Sol 129 368 2580 949440 serum free media 246 18000 4920 88560000 Air 10525 0 210500 0 H3PO4 6275 8.5 125500 1066750 NaOH 5892 15 117840 1767600 Pro. A reg buff 3174 10 63480 634800 Pro.A elution 5317 9.2 106340 978328 Pro A equil 11562 9.2 231240 2127408 ION eq buff 1730 11.2 34600 387520 ION wash buff 1732 22 34640 762080 IOX el buff 97 18.5 1940 35890 Nacl(1M) 1065 22 21300 468600 amm. Sulfate 74 480 1480 710400 polysorbate80 2 110 40 4400 sodium citrt dihydrate 0.25 2500 5 12500 Total raw material cost 98465716 19.2 Utilities Cost Table 19.2: Cost of Utilities Utilities requirement Annual requirement Rate (Rs./ unit) Cost per annum Cooling water 50000 Kg Rs. 15/m3 750 Electricity 58000 KW.hr Rs.8 /KW.hr 464000 Steam 80000 kg Rs.1.4/kg 112000 Total Utilities cost per annum (Rs.) 576750
  85. 85. 19. Total Cost of Production Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 80 Thus, Variable Cost = Raw Material Cost + Utility Cost = 990.42466 lakhs 19.3 Indirect cost Table 19:3 Indirect Product Cost Component Contribution Total Cost (Rs. In lakhs) Operating labour cost (OLC) 10 % of project cost 134.7429765 Repairs and maintainance 5 % of project cost 67.37148825 Supervision 15 % of OLC 20.21144648 Labour 15 % of OLC 20.21144648 Supplier 15 % of M & R 10.10572324 Indirect product cost (Rs. In lakh) 252.6430809 19.4 Fixed Charges Table 19:4 Fixed Charges Component Contribution Total Cost (Rs. In lakhs) Local tax 4 % of project cost 53.8971906 Insurance 1 % of project cost 13.47429765 Total fixed charges ( Rs. In lakh) 67.37148825 19.5 Plant Overheads Plant overheads = 40 % (OLC + Supervision + Repairs and Maintenance Cost) = Rs. 88.930 lakh Total plant Overheads (Rs. In lakh) = 88.930 lakh
  86. 86. 19. Total Cost of Production Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 81 19.6 General Expenses Table 19:5 General Expenses Component Contribution Total Cost (Rs. In lakhs) Administration cost 25 % of OLC 33.68574413 Distribution and selling 0.2 % of project cost 2.69485953 Generl expences ( Rs. In lakh) 36.38060366 19.7 Salary & Wages Salary and Wages = 9 % of variable cost = 0.09 x (990.42) Salary and Wages = Rs. In laks 1.0903/- Therefore fixed cost = Total indirect cost + Fixed charges + Plant Overheads + General Expenses + Salary and Wages = Rs. 446.415 lakh Fixed cost = Rs. 446.415 lakh. 19.8 Cost of Production Cost of production = Variable cost + Fixed cost Total cost of production =Rs1436.840592 in lakh. Now, We manufacture 10 kg of RITUXIMAB. Cost of production = Rs. 14368405.92=14370000/kg 19.9 Comments on Economic Feasibility Following is the production capacity of products and revenue generated from selling product: Products Rate ($/kg) Annual Capacity(Kg) Rate(Rs)/Kg total Cost(Rs) Product 300 10 18000000 180000000
  87. 87. 19. Total Cost of Production Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 82 Weighted average selling price of product = Rs. 1800000/kg Hence there is an earning of Rs. 3630000/kg and therefore Rs36300000 lakh / annum. Since the project is economically feasible. We can proceed to estimating the working capital and do further financial analysis of the project. 19.10 Summary Table 19.6 Summary of Production Cost Sr. No. Type Component Cost( Rs. in Lakh) 1 Variable Raw material 984.65 2 Utilities 5.76 Variable Cost of Production 990.42 3 Fixed Indirect cost 252.64 4 Total fixed charges 67.37 5 Plant overheads 88.93 6 General expenses 36.38 7 Salary and wages 10.90 Fixed cost of Production 446.40 Total cost of Production 1437.00
  88. 88. 20. Estimation of Working Capital Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 83 20. Estimation of Working Capital Working capital is the capital required to make the project work perform. The estimates are based on the guideline by Mahajani & Mokashi (2005). The following are the components of working capital. 20.1 Raw Materials We generally keep an inventory of 1 batch as storage of Raw Materials as work in progress. Steam is also generated in plant from waste heat boiler and air is compressed whenever required so no need to store it. The inventory, therefore, is not applicable for raw materials. 20.2 Product and Utility in stock The amount is estimated at the cost of production. Table 20.1 Product Streams as WIP and Utility costs for 1 batch Product No. of days Amount(kg) Cost(Rs)/Kg Total Cost(Rs) Product 15 0.5 12000000 6000000 0 Utilities 15 5 % of total 315789.4737 TOTAL PRODUCT COST ( Rs) 6315789.474 20.3 Maintenance Spares Calculating the plant and machinery item of project cost for 1 month. The total plant and machinery cost of the project = 673.7149 lakhs. A provision is made for maintenance spares as 0.1% of the plant and machinery item of the project cost for one month. Thus, maintenance spare inventory cost = Rs. 0.67374883 lakhs. 20.4 Other fixed costs These are estimated as 10% of the product inventory cost. Thus, other fixed expenses = 10% of (644) lakhs. = Rs. 6.31578947 lakhs.
  89. 89. 20. Estimation of Working Capital Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 84 Table 20.2 Total working capital break-up Parameter Cost(lakh) Raw material inventory cost 49.232858 Product inventory cost 63.1578947 Thus, maintenance spare inventory cost 0.67371488 Other fixed cost 6.31578947 TWC (Lac) 119.380257 20.5 Working capital source Following table shows source of total working capital. Table 20.3 Source of working capital Component Contribution Cost in lakh Borrowed WC 75 % TWC 89.53519 Margin money 25% TWC 29.84506427 Total Working Capital ( Lac) 119.3802571
  90. 90. 21. Estimation of Financial Expenses Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 85 21. Financial Analysis Financial Analysis involves estimation of financial expenses, depreciation values, key indicative ratios and the breakup of working capital as a function of time, all of which helps plan the execution and operations of the project. Table 21.1 Project Financing Components Total fixed capital investment 1347.43 Lac Debt : Equity 1.5:1 Term Loan 808.4579 lac is @12 % pa Equity participation 538.9719 lac Promotors contribution 274.8757 lac 51% Organizational participation 80.84579 lac 15% Public issue 183.2504 lac 34% Borrowed working capital 89.53519 lac is @ 16 % installment for 4 years 21.1 Estimation of Financial Expenses Term loan repayment period = 10 years; yearly in equal half annual installments Moratorium period = 2 years; after which equal installments are paid Working Capital Repayment Period = 4 year; annually in equal half annual installments Interest is found by computing simple interest for that period.
  91. 91. 21. Estimation of Financial Expenses Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 86 Table 21.2 Term Loan Scheduling Table 21.3 Borrowed Working Capital Repayment Schedule Installment Principle remaining Intrest payble Principle paid Installment paid Principle at year end Rs in Lac Rs in Lac Rs in Lac Rs in Lac Rs in Lac 1 89.54 7.16 8.42 15.58 81.12 2 81.12 6.49 9.09 15.58 72.03 3 72.03 5.76 9.82 15.58 62.21 4 62.21 4.98 10.60 15.58 51.60 5 51.60 4.13 11.45 15.58 40.15 6 40.15 3.21 12.37 15.58 27.78 7 27.78 2.22 13.36 15.58 14.43 8 14.43 1.15 14.43 15.58 0.00 Installment Principle remaining Interest payable Principle paid Installment paid Principle at term end Rs in Lac Rs in Lac Rs in Lac Rs in Lac Rs in Lac 1 808.46 48.51 0.00 48.51 808.46 2 808.46 48.51 0.00 48.51 808.46 3 808.46 48.51 0.00 48.51 808.46 4 808.46 48.51 0.00 48.51 808.46 5 808.46 48.51 31.49 80.00 776.97 6 776.97 46.62 33.38 80.00 743.59 7 743.59 44.62 35.38 80.00 708.20 8 708.20 42.49 37.51 80.00 670.70 9 670.70 40.24 39.76 80.00 630.94 10 630.94 37.86 42.14 80.00 588.80 11 588.80 35.33 44.67 80.00 544.13 12 544.13 32.65 47.35 80.00 496.78 13 496.78 29.81 50.19 80.00 446.58 14 446.58 26.79 53.20 80.00 393.38 15 393.38 23.60 56.40 80.00 336.98 16 336.98 20.22 59.78 80.00 277.20 17 277.20 16.63 63.37 80.00 213.84 18 213.84 12.83 67.17 80.00 146.67 19 146.67 8.80 71.20 80.00 75.47 20 75.47 4.53 75.47 80.00 0.00
  92. 92. 21. Estimation of Financial Expenses Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 87 21.2 Depreciation Schedule To estimate depreciation, the preoperative expenses and contingencies are distributed among the other project cost components, in their corresponding proportions. Following table contains the depreciation rates are used for the estimation of the working results. The rates of depreciation are fixed by the Income Tax Act and can be revised during the budget. Table 21.4 Depreciation Rates Depreciation rate Cost Component Value Depreciation rate Rs in Lac SLM (%) WDV (%) Land and site development ( L&D) 134.74 Building and Construction ( B& C) 107.79 3.34 10.00 Plant and Machinery (P & M) 673.71 10.34 25.00 Misc.fixed assets (MFA) 40.42 5.00 20.00 SLM: Straight line method. WDV: Written Down Value Method. In case of straight line method, the asset depreciates at a constant rate and the value after n years is given by (Mahajani & Mokashi 2005): Va  Vo  DSLM Vo  n Equation 23.1 In case of written down value method (also known as accelerated depreciation method), the value of asset at the end of any year n is given by: Va  Vo  1  DWDV n Equation 23.2 In the above equations: Va = Value of asset at the end of year n, Vo = original value, DSLM = Depreciation rate by straight line method, %. DWDV = Depreciation rate by written down value method, %.
  93. 93. 21. Estimation of Financial Expenses Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 88 The depreciation schedule is as follows: Table 21.5 Depreciation Schedule Year MFA B&C P&M Total SLM WDV SLM WDV SLM WDV SLM WDV 1 2.02 8.09 3.60 10.78 69.66 168.43 75.28 187.30 2 2.02 6.47 3.6 9.70 69.66 126.32 75.28 142.49 3 2.02 5.17 3.6 8.73 69.66 94.74 75.28 108.65 4 2.02 4.14 3.6 7.86 69.66 71.06 75.28 83.05 5 2.02 3.31 3.6 7.07 69.66 53.29 75.28 63.67 6 2.02 2.65 3.6 6.36 69.66 39.97 75.28 48.98 7 2.02 2.12 3.6 5.73 69.66 29.98 75.28 37.82 8 2.02 1.70 3.6 5.16 69.66 22.48 75.28 29.33 9 2.02 1.36 3.6 4.64 69.66 16.86 75.28 22.86 10 2.02 1.09 3.6 4.18 69.66 12.65 75.28 17.91 21.3 Sales Realisation Table 21.6 Sales Realisation Product Annual production Selling Price ( Rs/kg) Sales( Lac/Year) Rituximab 10 18000000 1800 21.4 Break-Even Analysis The following assumptions are made while carrying out the break-even analysis (Mahajani & Mokashi, 2005):  All expenses are bifurcated into fixed and variable costs.   The market conditions are ideal. Whatever is produced is sold immediately.   The selling expenses are fixed as some % of sales.   The following annualized (fixed) costs are not dependant on capacity utilization, namely, interest, charges on term loan, depreciation.   The equation used to estimate the break even capacity is:  S * X= V * X + F Equation 23.3
  94. 94. 21. Estimation of Financial Expenses Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 89 where, X = Break-even capacity S = Price of one ton of product V = Variable cost of production per ton F = Fixed cost of production = Interest on Term Loan + All Overhead costs Therefore, X = F/(S-V) Equation 23.4 The fixed cost of production includes the overheads, administrative expenses, average financial expenses and depreciation (SLM). Substituting the required values, F = 326.08 lakh/ annum V = Similarly, the sale price for products in the above proportion can be calculated as: S= n X= 0.004027ton/ annum X =40.28 %.
  95. 95. 21. Estimation of Financial Expenses Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 90 21.5 Ratio Analysis 21.5.1 Performance Ratios A. Payback Period Assuming 100 % capacity utilization Table 21.7 Payback Period Gross cost of production 143700000 Rs. annual sales 180000000 Rs. Gross profit 36300000 Rs. Total capital investment 130222198.1 Rs. Payback period 3.587388378 Years 3 Year and 7months B. Return on Investment (ROI): Assuming 100% capacity utilization, ROI is given by, Gross Profit * 100 Equation 21.5 Total Capital Investment Return on Investment = 27.87 %65.78% C. Profit Margin: Assuming 100% capacity utilization, Profit margin = ( Gross profit / Sales) * 100 Equation 21.6 Profit margin = 20.16 % D. Net Assets Turnover: Net assets turnover = (Sales / Project cost) Equation 21.7 Net assets turnover = 1.33
  96. 96. 21. Estimation of Financial Expenses Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 91 21.5.2 Financial Ratios A. D:E Ratio Debt : Equity = 1.5:1 B. Interest Cover: 100% capacity utilization is assumed and interest cover is calculated for the first year. Interest Cover = Gross profit / Interest Equation 21.8 Interest Cover = 3.26 21.6. Estimates of Working Results The projected working results for 10 years are presented in tabulated form. A dividend of 20% is assumed from the first year. Following are the terms involved in this evaluation:  Gross Profit = Sales – Gross Cost of Production  Operating Profit = Gross Profit – Financial Expenses – SLM Depreciation  Taxable Profit = Gross Profit – Financial Expenses – WDV Depreciation – Loss from previous year (if any)  Corporate Tax = 30% of Taxable Profit  Profit after Tax = Gross Profit – Financial Expenses – Corporate Tax  Profit for Dividend = Profit after Tax – SLM Depreciation  Dividend = 20% of Profit for Dividend  Profit after Dividend = Profit for Dividend – Dividend  Net Cash Accruals = Profit after Dividend + Depreciation(SLM)
  97. 97. 21. Estimation of Financial Expenses Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 92 Table 21.8 Estimate of working capital for years 1-5(All figures in Rs. (Lakhs), except capacity and utilization) Year 1.0000 2.0000 3.0000 4.0000 5.0000 Capacity utilisation 70.0000 70.0000 80.0000 80.0000 90.0000 % capacity 0.0070 0.0070 0.0080 0.0080 0.0090 Sales 1260.0000 1260.0000 1440.0000 1440.0000 1620.0000 Gross cost of production 1005.9000 1005.9000 1149.6000 1149.6000 1293.3000 Gross profit 254.1000 254.1000 290.4000 290.4000 326.7000 Financial expenses 97.0149 97.0149 95.1255 87.1073 78.0981 Depriciation ( SLM) 75.2836 75.2836 75.2836 75.2836 75.2836 Depriciation ( WDV) 120.2100 115.0700 108.6452 83.0523 63.6748 Operating profit 81.8015 81.8015 119.9909 128.0091 173.3183 Taxable profit 36.8751 42.0151 86.6293 120.2404 184.9271 Corporate profit 11.0625 12.6045 25.9888 36.0721 55.4781 Profit after tax 146.0225 144.4805 169.2857 167.2206 193.1238 Profit for devident 70.7389 69.1969 94.0021 91.9370 117.8402 devidend 14.1478 13.8394 18.8004 18.3874 23.5680 Profit after devidend 56.5912 55.3576 75.2017 73.5496 94.2721 Net cash accural 131.8748 130.6412 150.4853 148.8332 169.5557 Table 21.9 Estimate of working capital for years 6-10 (All figures in Rs. (Lakhs), except capacity and utilization) Year 6.0000 7.0000 8.0000 9.0000 10.0000 Capacity utilisation 90.0000 95.0000 95.0000 100.0000 100.0000 % capacity 0.0090 0.0095 0.0095 0.0100 0.0100 Sales 1620.0000 1710.0000 1710.0000 1800.0000 1800.0000 Gross cost of production 1293.3000 1365.1500 1365.1500 1437.0000 1437.0000 Gross profit 326.7000 344.8500 344.8500 363.0000 363.0000 Financial expenses 67.9754 56.6015 43.8218 29.4625 13.3284 Depriciation ( SLM) 75.2836 75.2836 75.2836 75.2836 75.2836 Depriciation ( WDV) 48.9825 37.8241 29.3333 22.8580 17.9073 Operating profit 183.4410 212.9649 225.7446 258.2539 274.3880 Taxable profit 209.7421 250.4245 271.6950 310.6795 331.7643 Corporate profit 62.9226 75.1273 81.5085 93.2038 99.5293 Profit after tax 195.8020 213.1212 219.5197 240.3337 250.1424 Profit for devident 120.5184 137.8376 144.2362 165.0501 174.8588 Dividend 24.1037 27.5675 28.8472 33.0100 34.9718 Profit after dividend 96.4147 110.2701 115.3889 132.0401 139.8870 Net cash accrual 171.6983 185.5537 190.6725 207.3237 215.1706
  98. 98. 21. Estimation of Financial Expenses Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 93 21.7 Discounted Profit Flow Analysis A. Weighted Average Cost of Capital The weighted average cost of capital is given by: WACC Di T Er E Equation 21.9 D  E For our project, D:E = 1.5:1 iT= 0.12 rE= 0.30 (assume). We have WACC = 0.192 = 19.2% B. Hurdle Rate of Return (K): K= 22.5% (> WACC) C. Present Value of Profit (PV) The present value of profit is given by: ∑ Equation 21.10 where, Pn denotes the Gross Profit Projection for the nth year. Substituting the values, we have, PV = Rs. 1670.986746 lakhs. D. Present Value of Investment The total capital investment is Rs. 7493 lakhs. Out of these, assume that Rs. 6000 lakhs are spent in the first year of investment and the rest are spent in the second year. It takes two years for the construction of project.
  99. 99. 21. Estimation of Financial Expenses Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 94 Present Value of Investment = Io  (1  K ) I1I2   = Rs.1414.92975akhs. E. Net Present value of Profit The net present value of profit is given by: ∑ Equation 21.11 = 256.056 Lakhs The project thus seems attractive. F. Profitability Index: The Profitability Index is given by: ∑ Equation 21.12 PI = 1.18 G. Internal Rate of Return: The Internal rate of return (IRR) is the value at which the NPV is zero. It is given by: ∑ Equation 21.13 IRR = 52.2% As IRR is greater than WACC, project is attractive.
  100. 100. 22. Storage, Utilities and Effluent treatment Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 95 22. Storage, Utilities and effluent treatment 22.1. Storage The entire process demands high level of monosepsis. Hence both the raw materials and the products need to be stored in extremely sterile environment. The raw materials are mostly biological grade materials and are stable at room temperature. The product cannot be exposed to high temperature. In order to retain its activity for a long time it is stored at -25°C. 22.2. Utilities The utilities required in the plant are 4 bar saturated steam ,cooling water and electricity Amount of steam required for sterilization of Bioreactor =2280+11+5.5≈3000kg.Besides steam is also required to sterilize the pipelines and other equipment. Hence total steam requirements is 4000kg Cooling water is consumed during sterilization and the fermentation process. Total cooling water required =214788+520+1042+5.5×3600×85=1900tonnes≈1900 m3 .Besides water forms a major portion on the fermentation medium too. Cooling water is re-circulated and so for annual requirement we only need to take consideration losses of evaporation and other losses. Let, cycle time of 1 day. So water required per batch is 126.66 TPB. Water losses = M = 1.73 ×0.001 ×∆T × Vc × (Kc/Kc-1) Water losses per batch are = 2.5 Ton Annual fresh water required is 50 Ton

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