DESIGN A PLANTTo 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 INDEX 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 Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB INDEX 23 Safety ,Health and Environment 97 24 Conclusions 100 25 References 101 26 Appendix A 103 Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Antonio Grillo-López.1. Introduction 1. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB. This document is a project feasibility analysis for manufacture of 100000. this drug could either completely treat or extend the lifetime of the patient. along with several colleagues. Manufacture of 100000. This extraordinary feat had very humble beginnings. 1. Depending on the severity of the cancer. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 1 . Introduction. pioneered a new drug named rituximab that serves as the first FDA approved antibody to treat cancer. 1. Many have tried. He.Rituximab Finding a cure for cancer has always been a goal for many health care professionals. but few have made as much of a stride as Dr. sodium chloride (9 mg/mL) and Water for Injection. and apoptosis. clear.35 mg/mL). activate the complement. 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.7 mg/mL). Gentamicin is not detectable in the final product. The pH is 6.2. Antibody-antigen complexes. Introduction A Rituximab is a genetically engineered chimeric murine/human monoclonal antibody consisting of a glycosylated IgG1 kappa immunoglobulin with murine light. Directed against the CD20 antigen. Rituximab is a sterile. ADCC is a process where the antibody-antigen complex forms and then attracts other components of the immune system. such as the one between rituximab and CD20.Mechanism of Action Rituximab kills cancerous B cells via three main mechanisms: complement-dependent cytoxicity (CDC). Rituximab is produced by mammalian cell (Chinese Hamster Ovary) suspension culture in a nutrient medium containing the antibiotic gentamicin. 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. including natural killer cells. The natural killer cells also carry granules filled with cytotoxic molecules. The granules can also destroy the cells by attacking the nucleus. antibody-dependent cell-mediated cytoxcity (ADCC).and heavy-chain variable regions (Fab domain) and human kappa and gamma-1 constant regions (Fc domain). The final mechanism is known as Manufacture of 100000. The product is formulated in polysorbate 80 (0. When the granules are released after the natural killer cells bind with rituximab. CDC occurs when a large group of plasma proteins (complement) work together to destroy invading pathogens and malignant cells. 1.5. The receptors on these cells recognize and bind to the tail of the rituximab antibody. 1. sodium citrate dihydrate (7. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 2 . Rituximab is supplied at a concentration of 10 mg/mL in either 100 mg/10 mL or 500 mg/50 mL single-use vials. preservative-free liquid concentrate for intravenous administration. colorless. The cytoskeleton collapses upon itself. 1. whether these mechanism act independently or in concert.Properties Sr.5 10 Binding affinity for CD20 8. 1. Despite this. It is still unclear.0 nM antigen 11 Solubility in Water Soluble Manufacture of 100000. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 3 . the cell has effectively destroyed itself. it signals the cell to start the process. Property Value No. rituximab still proves to be an effective cure. or programmed cell death. Introduction apoptosis. the nucleus condenses.3. however. 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. At the end of apoptosis. When rituximab bonds to CD20 and forms the antibody receptor complex. Apoptosis is defined the death of cells that occurs as a normal and controlled part of an organism’s growth or development. and the DNA fragments into small pieces via enzymes. Membrane-bound vesicles are also shredded.7 g/mol 8 Boiling Point(degrees C) 100 9 Optimal pH range 6. Reddy’s Laboratories Ltd.(INDIA) 2 Genentech .000 lymphoma patients are cured. IMPACT The impact that rituximab had is unprecedented. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 4 . South San Francisco. It provides a cure for curable lymphomas such as diffused large cell lymphoma. something that can be replicated by any ordinary person with persistence and determination. Each year. Introduction 1. around 50. Since its introduction in 1997 until 2010. The lifetime of patients with incurable lymphomas have been extended.7 billion. Geduld led to his success. himself. there has been a long of stagnation in finding cures or ways to extend lifetimes.4. Antonio Grillo-López will inspire others to follow in his footsteps. He.5. Hopefully the success of Dr. It has been considered the top anticancer drug in the world since 2001. 1. has said that he is neither a saint nor a magician. Manufacturers 1 Dr. over two million patients have been treated. Manufacture of 100000. Recent numbers further demonstrate the success of rituximab. Sales in 2010 alone totaled $6. Prior to this discovery. 1. Project Site: Raigad.A elution 5317 9. A reg buff 3174 10 31740 Pro.Maharashtra Raw Material Requirements: Annual Raw material Batch req ( kg) Rate (Rs/kg) cost(Rs) Inoc.5 NaOH 5892 15 88380 Pro.4 Pro A equil 11562 9. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 5 .4 ION eq buff 1730 11. Executive Summary Product: RITUXIMAB Installed Capacity: 100000 vials of 10 ml each with strength of 10 mg/ml.5 1794. 2.2 19376 ION wash buff 1732 22 38104 IOX el buff 97 18.25 2500 625 Total Raw material cost 4923286 Manufacture of 100000. Executive Summary 2.5 Nacl(1M) 1065 22 23430 ammonium Sulfate 74 480 35520 polysorbate80 2 110 220 sodium citratet dihydrate 0. State.2 48916. Media Sol 129 368 47472 serum free media 246 18000 4428000 H3PO4 6275 8.2 106370. Project Type: Greenfield.5 53337. 2. Return on Investment: 65.84lakhs (15% of equity).5:1. Promoter’s contribution: 274. Public Issue: 183. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 5 .2 lakhs Debt: Equity Ratio: 1. Project Evaluation: Break-Even Capacity: 40. Executive Summary Gross Cost of Production: 1437 Lakh of product produced.2%.6 % of installed capacity.) Payback Period: 3 year 7 months. Financing: Total Fixed Capital Investment: 1302.25 lakhs (34% of equity). Term Loan: 808.87 lakhs (51% of equity) Organizational participation: 80.28% (= 59.18 Internal Rate of Return: 52. Sales: Selling Price (Average): 18000000 Rs/kg of Rituximab Total Sales: 1800 Lakh/ annum.76 % Manufacture of 100000.45 lakhs @ 12 % per annum. (Desired return on equity = 30%) Profitability Index: 1.41 % Weighted average cost of capital: 19. 3.Process Selection 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. Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 7 3.Process Selection 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 . Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 8 4.Process description. 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. Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 9 or synthetic origin.22 µm vacuum filter (vaccucap 90): Gelman science #pn 4622-filterling capacity up to 10 L Manufacture of 100000.1.1. chemically-defined medium optimized for the growth of Chinese hamster ovary (CHO) cells and expression of recombinant proteins in suspension culture. DMEM and buffer preparation. We import the CHO medium from Life Technologies. 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. as well as no undefined lysates or hydro lysates. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 10 . CD CHO medium contains no proteins or peptide components of animal.W.) ▪ 0.1.Process description. With a proven track record of quality for more than 10 years. plant. Medium preparation: CD CHO medium is a protein-free. 4. 4. serum-free. 500 g (cell culture grade) ▪ 3X Distilled Water (D. Batch reactor CHRTGPH Reaction-2 CHRTGPH-2 CHRTGPH-3 Reaction-1 Reaction-3 Filtration Y-3 4. 4) Freeze and store. Always aliquot and freeze serum. ▪ DPBS (Dulbecco's PBS): sigma #D 5652.Process description. 100 ml ▪ FBS (Fetal Bovine Serum): JBI #S 001-01. To heat-inactivate serum 1) Thaw the frozen serum (company supply FBS in -20℃) at 37℃ for 5-6 hours. and add it to medium just before use. 10 bottles/pack. but our system use fetal Bovine serum (FBS/FCS). 1 bottle for 1 L ▪ Trypsin-EDTA (10X): sigma #T 4174. . 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. So. b) FBS Serum supplies growth factors and nutrients. These are the serum variables you must consider. (Caution !!) Serum is very expensive. where it will be fine for Manufacture of 100000.Heat-inactivate serum: Serum in subjected to heat to inactivate components such as complement. . Thaw aliquots in a 37℃ water bath as needed. Serum requirement is dependent on cell type. Some cell lines have been 'trained' to survive in medium with low serum. 4. Store unused portions of thawed aliquots in the refrigerator.The percentage of serum: Most cells require 5-20% in the medium for good growth (all of our cell line require 10% FBS). must check how much serum amount your cells need before starting cell culture. . 500 ml. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 11 .The types of serum: Some cells like horse serum or calf serum. W. The complex media contain a large number of different amino acids. 4. 8) Store media at 4℃ in the dark until use. 1) Measure out 5% less 3X D. Stir until dissolved (Do not over mix).Process description. Adjust pH of media to 0. additional vitamins and Extra metabolites in F12 (optimizing by cloning) and in Dulbecco's modified Eagle's MEM (DMEM) [Dulbecco. 5) Add D. 1959]. 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. CMRL 1066 [Paker. MB752/1 [Waymouth. 6) The final pH of the media should be pH 7. 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. than desired total volume of media. The pH units will increase 0. Add slowly With stirring. DMEM-F12 [Barnes and Sato.W.3 upon filtration.2-0. and Ham-F12 [Ham.3 below the Desired working pH (~ 6. 1965].95) using 1 N NaOH or 1 N HCl (generally HCl).4.W. RPMI 1640 [Moore. 1980]. After pH has been adjusted. 1959]. 1957]. 1967].7 g of NaHCO3 to make 45mM concentration/L of media. and add it to media. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 12 . . at RT with gentle stirring (Do not over-stir or heat). 1950].W. 4) Add 3. 1950] - Complex to Medium 199 [Morgan. 3) Rinse remained powder in the package with D.1-0. to desired volume. Manufacture of 100000. 2) Add powdered media to D. 2 CHO K1 (+) Media This media is suitable for CHO K1 cells which stably express fluorescent proteins and are resistant to geneticin. 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. These bottles should be washed with bleach and water (3x with bleach and 9x with water). To prepare the media you will need the following: 1.400 mL of DMEM – Low Glucose 2.2. 430 mL of DMEM 2.2.50 mL of FBS Manufacture of 100000.1 CHO K1 (-) Media This media is suitable for non-transfected CHO K1. 10 mL of L-glutamine Mix all reagents together and run through a sterile filter. 4. 5 mL of Pen/Strep 4. After being autoclaved you can spray the outside with EtOH and bring into the laminar flow cabinet. 5 mL of Non-essential amino acids 5. Media for Cell Culture We are now using washable and reusable glass bottles to prepare our media and our PBS solutions. To prepare the media will need the following: 1. or transiently transfected CHO cells which do not have any antibiotic resistance.Process description. then dried and autoclaved. 50 mL of heat inactivated FBS 3. 4. 4. The media bottles have tops which are compatible with the disposable sterile filters from VWR. 4.2. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 13 . 4.Process description. 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 0C. 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. Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 14 4.Process description. 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: Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 15 4.Process description. 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. Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 16 All kinetic data were submitted to curve fitting techniques. 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. was calculated using the following equation: Manufacture of 100000. The derivative with respect to time was then calculated for the values obtained with the help of the polynomial function. 5. µ. Kinetics and thermodynamics of process 5. glucose is taken as the limiting reactant. The specific consumption or production rates could then be calculated by dividing the derivative by the viable cell concentration (Xv) at selected time points. Kinetics and thermodynamics of the process 5. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 17 .1. In this case. The apparent specific growth rate. An appropriate polynomial function was fitted by the least squares method to the measured concentration data. Fig. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 18 . Kinetics of CHO cell growth. Hence proper conditions and control is required for maximum yield. 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 (_).1. 5. in 96 well plates containing 200 mL of different media: serum-free reference medium (*). Experiments were performed in triplicate Manufacture of 100000. Below the growth trend and substrate consumption for the CHO is given. Kinetics of CHO cell growth. serum-free medium supplemented with 25 mg/L of sinapic acid (&).. Kinetics and thermodynamics of the process 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. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 19 . 5.2. But we can safely conclude that the overall reaction is thermodynamically feasible. Kinetics and thermodynamics of the process 5. Thermodynamics Biochemical pathways are complicated and it is not possible to find the ΔG values for every reaction taking place. Manufacture of 100000. 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 20 . 6. Process Block Diagram Nutrient Medium Inoculum Preparation Bioreactor Centrifugation Protein A Ion Exchange Chromatography Chromatography Air Cell mass Diafiltration sterilization Air VR filtration Storage VIALS FILLING & Final Filtration Formulation PACKING Manufacture of 100000. Hence the manufacturing site should be close to metro cities so that transportation cost is reduced 7. These have effect on the fixed cost of the plant. 7. units and equipments.1. 7. Market for the Product: The product Rituximab is used for cancer treatment.1. This land must be available at affordable price as this would eventually be contributing to the fixed cost of the project. Land: Adequate land space must be available for all the buildings. 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 21 .1. So it is important to consider this component in site selection.3. Also provision for any future growth has to be considered.1 . Manufacture of 100000. and identification of seismic zone and height of water table. minimum and maximum temperature.4.Availability of raw materials: Raw material contributes a major share to the operating cost of the project. Site Selection Proper site selection is one of the factors that govern the success of any project.1.2. 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. Proper soil survey to avoid the mechanical breakdown of the plant is necessary.1. This in turn is going to determine the cost of production of the product eventually deciding the profit margin. 7. Climatic conditions: The necessary climatic data like average rainfall. its bearing capacity. 1.5. Site Selection 7. frequency of cyclones and hurricanes should be considered while selecting a site. Profitability factors 7. Soil Assay: This includes the type of soil. 7. Other space requirements like effluent treatment plant or green belt also should be given due consideration. 8. 7. Manufacture of 100000. Environmental considerations: Having a common effluent treatment plant in an industrial complex always helps reduce the load on the project. 7.3 Infrastructure: The existence of well developed infrastructure is desirable.7 .2 Labour: Availability of skilled labour is important.2. 7. e-mails. 7.1.2. Also general civic amenities should be easily accessible.1.Water quality and availability: This is the most important parameter for site selection since water is required in huge quantities for the process. 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 22 . Choosing a site which guarantees the uninterrupted supply of water having compatible qualities at affordable rate is desired.1 Communication: Communication facilities like Telephone.2.2. 7. etc. 7. Telex. The site should easily accessible by road and railways.1. Water must be available in adequate amounts and with good quality at cheaper rates. Productivity factors 7.Power: Regular and uninterrupted power supply at conceded rates is an essential factor to run the project smoothly. Site Selection 7.6 . should be there at the site location. Fax. 3.3. 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.4.Site evaluation Based on the above mentioned factors the two sites which seem suitable to set up the manufacturing unit are Hosur. 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. 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 23 . 7. There should be proper utilization of resources.1 Appropriate Utilization of Raw materials: The natural resources of the country have to be utilized appropriately and efficiently. Site Evaluation Criteria Site A Site B Land and site development 8 7 Building and civil 7 7 construction Climate 8 8 Raw Material source 8 10 Product Market 6 9 Manufacture of 100000.3 Security of the Nation: The site should not be situated in a politically sensitive area. 1.3.Both of these sites have been declared as biotech SEZ’s by the respective governments. Table 1. decreasing the load on the public transportation system.3 Prosperity factors 7. 7. Site Selection 7. 7. so it does not endanger national security. Krishnagiri district under TIDCO(Tamil Nadu Industrial Development corporation) (Site A) and Raigad under MIDC(Maharashtra Industrial Development corporation )(Site B).2 Dispersal of Industries: Utilization of most of the available land. Site Selection Water/Effluent treatment 8 6 Power 6 8 Environmental Consideration 8 6 Housing and social 7 8 community factor Staff transport 5 8 Equipment transport 8 9 Taxes and subsidies 7 8 TOTAL 86 92 Therefore Raigad. Manufacture of 100000. 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 24 . Maharashtra is chosen as the manufacturing site. 7. The production bioreactor operates under a fed batch mode. Material balance A] Inoculum preparation The inoculum is initially prepared in 225 ml T-flasks. B] Bioreaction section Serum-free low-protein media powder is dissolved in WFI in a stainless steel tank (MP-103). The fermentation time is 12 days. A stirred-tank bioreactor (PBR1) is used to grow the cells. Sterilized media is fed at the appropriate amount in all of these four initial steps (3. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 25 . 175. 8. 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.5 kg of product. The broth is then moved to the first (25 L) and second (50 L) seed bioreactor. 11. For the seed bioreactors the media powder is diluted using WFI in one prep tank. The concentration of media powder in the initial feed solution is 11.73 g/L. The material is first moved to roller bottles (25 L).6.5 kg Rituximab Manufacture of 100000.2 µm dead-end polishing filter (DE-103). The volume of broth generated per bioreactor batch is approximately 50 L. which contains roughly 0. which produce the therapeutic monoclonal antibody (Rituximab). Material Balance 8. then to 50 L and subsequently to 75 L disposable bag bioreactors.000 vials) So 10 Kg to be produced in 300 days Total batches = 20 1 batch production = 0.4 kg/batch respectively). The solution is sterilized using a 0. Basis: Per batch Total working days = 300 days Total Product to be produced= 10 Kg (10mg/ml 10ml fill 100.6. 43.4. 1.2 × 25=105 g.5g.73×V = 20×25=293.5g = 1. = 11.73×50=586. Bioreactor B001 Volume = 25 Liters Product Con = 11.73 g/l/day Total mass of cell = 11. 8.76 g The size of B001 is 34 Lit with 25 L working volume.5 1 Serum free media 0 0. Material Balance 8.2. Total mass of cell in 1 batch ( 6 days) =6000 mg = 6 g Manufacture of 100000. Serum free media supplement 4.75g Total mass of cell in 1 batch ( 6 days) =1762. Bioreactor B002 Volume = 50 Liters Product Conc.73 g/l/day Total mass of cell = 11.73×V = 11.2 g/lit Total serum free media supplement = 4. 1 B001 3 2 2 Table 8. 1:Across B001 input(kg)(1) input(kg)(2) output(kg)(3) Cell mass 0.105 0 8. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 26 . The concentration of media powder in the initial feed solution is 42 g/L. Serum- free low-protein media powder is dissolved in WFI in a stainless steel tank. The volume of broth generated per bioreactor batch is approximately 80 L. 8.065 m3 with 0. 3 B002 5 4 2 Table 8. 2: Across B002 input(kg)(3) input(kg)(4) output(kg)(5) Cell mass 1× 10-3 0 1.2 g/lit Total serum free media supplemet =4. which contains roughly 50 kg of product. The solution is sterilized using a 0. Serum free media supplement 4.2 µm dead-end polishing filter. Manufacture of 100000. Material balance across the Production Bioreactor B003 The size of the production Bioreactor B001 is 0. The production bioreactor operates under a fed batch mode.05 m3 working volume.5 × 10-3 Serum free media 0 210 0 8. Material Balance The size of B002 is 65 Lit with 50 L working volume. 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.2 × 50=210 g. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 27 .3. which produce the therapeutic monoclonal antibody (Rituximab). A stirred-tank bioreactor is used to grow the cells. The fermentation time is 12 days. 61 0. 0.3: Across B003 input(kg)(5) Input(kg)(6) output(lit)(7) Amino Acid(20) 0.035 2 Vitamin (9) 3.75× 10-3 Serum free media 0 0. Amount of media required for composition = 0.8039kg of media needs to be supplied. COMPONENT mg/L(x) Conc (75*x) Kg 1 Amino Acid(20) 476.75 g Amount of serum free media=4. =0. Material Balance 5 B003 7 6 2 Total cell mass=75 ×11. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 28 .75g=879.622 0 0 Cell mass 0 879.4 0.315Kg.2×75=315g.73=879.9 0.035 0 - Vitamin (9) 270 × 10-6 0 0 Organic Compound(8) 0.315 Total volume(lit) 0 0 72 Manufacture of 100000. 0.14hrs.6 270 × 10-6 3 Organic Compound(8) 1948.8039 Table 8.622 Total .NO. SR.146 0 0 Inorganic Salts 0.8039 Time taken for it to get completely consumed Thus after every 7.146 4 Inorganic Salts 8300. 8. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 29 .2 μm dead-end filters to ensure sterility.3.48 98 8. 8. During this step.5 h with Polysorbate 80 to inactivate viruses (in P-22 / V-111). roughly 2% of Mab is lost in the solids waste stream resulting in a product yield of 98% for 3.78 1.24 826. Material balance across the Centrifuge C001 Between the downstream unit procedures there are 0. The generated biomass and other suspended compounds are removed using a Disc-Stack centrifuge.3 70. The yield on product is 98% and this is represented by the product denaturation feature of the Diafiltration operation. The yield on Mab for this step is 90%. The concentrated protein solution is then chemically treated for 1. Manufacture of 100000. 7 C001 9 8 Table 8. Material Balance 8.97 15.8 hrs.The protein solution is then concentrated 5x and diafiltered 2x (in P-21 / DF-101).73 98 Volume(Lit) 71.4.4: Across C001 input(7) output(8) output(9) % yield Mass of cell (g) 841. 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). 22 95 Volume(Lit) 70.52 68. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 30 .5.5: Across F001 input(9) output(10) output(11) % yield Mass of cell (g ) 826.28 800.22 95 8. 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. 11 F002 13 12 Manufacture of 100000. Material Balance F001 11 9 10 Table8. 20% of Mab is lost during the HIC procedure. 8. 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%.48 3.73 41. 69 60. Material balance across Difiltration F004 15 F004 17 16 Manufacture of 100000. 8.27 6.6.44 78.27 90 8.6.96 6.35 90 Volume(Lit) 60. Material Balance Table8.6: Across F002 input(11) output(12) output(13) % yield Mass of cell (g ) 785.02 54.96 90 Volume(Lit) 66. Material balance across Virus Retentive Filtration F003S 13 F003 15 14 Table8.47 706.96 70.7: Across F003 input(13) output(14) output(15) % yield Mass of cell (g ) 706.25 90 8.61 636. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 31 . 78 604.71 51. Material balance across Formulation Column FC001 17 3 FC001 18 17 10 P002 12 13 10 P002 13 21 12 19 10 P002 13 12 20 10 P002 13 12 Table 8.97 0 Sodium Citrate dehydrate(18) 399.8: Across F004 input(15) output(16) output(17) % yield Mass of cell (g ) 636.25 2. Material Balance Table8.592 Manufacture of 100000. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 32 .56 0. 8.47 0 Sodium Chloride(19) 488.54 95 8.35 31.25 0 Cell mass(20)(g) 604. 9: Across FC001 input(gm) output(Kg)(20) Polysorbate-80(17) 37.7.56 95 Volume(Lit) 54. 64 Manufacture of 100000.50 0.5 99 Volume(Lit) 50.8.11 : 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. Material Balance 8. Vials Filling Table8. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 33 .8.36 5. Summary of the entire process The material balance summary across all the units in the process is as follows Table8. 8.10: Across V001 input Output Output % yield Mass of cell (g ) 592.50 50 99 8.86 586. Energy balance 9. The heating cycle The vessel is heated from room temperature to 121°C by using saturated steam at 4bar pressure. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 34 .1.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. Thereafter.1.1. it has to be heated to 121°C. be held at that temperature for a certain length of time and cooled to 37°C. Energy balance 9.2. Across production Bioreactor B003 For sterilizing the Bioreactor and the Bioreactor media. fermentation being an exothermic process the temperature has to be maintained at 37°C by passage of cooling water through the coils 9. The properties of steam at 4 bar are : temperature difference:121-30=91°C=365 k Cp: Specific heat capacity of Bioreactor media: 4. the temperature at which fermentation takes place.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 9.18 kJ/kg : difference in cooling water inlet and outlet temperature:5°C :temperature difference of the Bioreactor media:121-37=84°C Manufacture of 100000. 9.1. 1. 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.The heat removal rate should be 115 kW.5 kg/s 9.000kJ/kg. The metabolic heat generation rate is assumed to be 10 kW/m3 (Biotranformation and bioprocess. flow rate of cooling water required is 5. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 35 . 9. Energy balance 9. Manufacture of 100000.3.5.Maintaining of Bioreactor temperature at 37°C Bioreactor is an exothermic process. Therefore.pg 182). operating conditions (namely. and physical and chemical properties of the substrates and the microbe. Considering the difficulty of controlling and maintaining monosepsis condition in a continuous mode. Process Design of Bioreactor 10. 10. fed batch mode is used. Equipment design 10.2. Hence less number of impellers should be used Manufacture of 100000.1. Bioreactor geometry (Walas.3. 10. Equipment Design 10.075 m3. Bioreactor type The type of Bioreactor depends on the nature of the process (including cell kinetics).1. 10.1990.1. volume of substrate media necessary in the bioreactor is 0. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 36 . Pg no . mode of operation and gas liquid flow patterns).1.For this scale of operation a mechanically stirred tank reactor consumes less power per unit volume as compared to non-mechanically agitated system. Since it is aerobic fermentation. Mode of operation As can be seen in Section of material balance intermittent supplies of media is required to maintain the cell growth composition. For the required production capacity.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.1. continuous supply of oxygen is essential. the air flow rate for optimal production is found to be 1 vvm (El Enshasy . For pilot scale At this calculated .37 ( ) 6.4. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 37 . is 1.1.515W s Manufacture of 100000. et al. 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. Hence we use a mixed flow pitched blade turbine impeller.H.2008) ( ) During scale up . However they consume high power. Equipment Design 10.the objective is to maintain the same mass transfer coefficient of oxygen. 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. 10. Manufacture of 100000. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 38 .5.Sparger design Gas hold up in the system (Handbook of chemical engineering calculations. Pg no.1. Equipment Design For production scale ( ) s 10.2 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. 575) ( ) = volumetric flow rate of air = 0. 10. Native Protein A – immobilized Protein A is ideal for polyclonal IgG purification. The 46.7kDa-protein consists of a single polypeptide chain that is essentially devoid of carbohydrate. providing a binding capacity greater than 34mg human IgG/mL resin (approx.2. Properties of crosslinked 6% beaded agarose (CL-6B): Support pH Stability: 2 to 14 (short term).6). 3 to 13 (long term) Average Particle Size: 45 to 165 microns Exclusion Limit: 10.000. 16 to 17mg mouse IgG/mL resin) Manufacture of 100000. the most popular resin for protein affinity purification methods. IgG- binding function is optimal at pH 8. Equipment Design 10. 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. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 39 .000 to 4. 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. 1mL/minute. leach-resistant covalent bonds.) Inert and stable – superior manufacturing method immobilizes Protein A by charge-free. Maximum Linear Velocity: 30cm per hour Maximum Pressure: less than 1. 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.5 bar. but efficient binding also occurs in neutral and physiological buffers (pH 7. Agarose resin – support is crosslinked 6% beaded agarose (CL-6B). 10.2.000 daltons Maximum Volumetric Flow Rate: approx.Protein A CHROM COLUMN Protein A is a cell wall component produced by several strains of Staphylococcus aureus.0 to 7. complete purification kits. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 40 . Manufacture of 100000. Uses permeable membrane to separate the components mainly based on size and column-based gel filtration. 10. replace or lower the concentration of salts or solvents from solutions containing biomolecules.3. Process Design of Diafiltration Unit Is a technique that uses basic principles of filtration to completely remove. two sizes of FPLC-ready chromatography cartridges. Equipment Design Multiple formats – choose from bottled resin slurries. centrifuge-ready columns. and 96- well filter plates. 10. direct) Direct Flow: . Flux rate decreases as volume filtered increases Manufacture of 100000.3. Large molecule trapped on membrane and forms gel . More susceptible to fouling .1. Design Consideration a) Type of flow (tangential vs. Equipment Design 10. 10. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 41 . 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. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 42 .Typical MW of mAb: 150kDa => 30000 MWCOO. Equipment Design Tangential Flow . Solute diffuses through the surface of the membrane tangent to the flow of the feed .. Reference:-Millipore Inc. c) Type of diafilter modules i) Flat sheet tangential flow ii) Hollow fibre iii) Tubular iv) Spiral wound Manufacture of 100000. Minimize buildup of molecules – less fouling .For protein separation: 30 LMH. 10. Prevents rapid decline in flux rate. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 43 .requires pumps Discontinuous Concentration and dilution cycles Usually more feasible on a laboratory scale Manufacture of 100000. 10. 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). Equipment Design d) Continuous vs. More suited for process scale. 49 L/min -UNIT NUMBER: Stainless stain housing Manufacture of 100000.2. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 44 . area = filtrate vol / (filtrate flux * process time) = 50/(30x2)=0. Equipment Design Effect of diafiltration volume on membrane area requirement and percent of acetic acid removal 102 29 100 27 Percent of Acetic Acid removal 98 Membrane Area (m2) 25 96 23 94 21 19 92 17 90 15 88 0 1 2 3 4 5 6 Diafiltration Volume Membrane Size Removal of Acetic Acid 9. 10.83=2.83 m2 Pump Feed rate (L/min) = Feed flow rate X Area = 3 X 0.2. Final design Feed flow rate = 3L/min/m2 -Hollow fiber cartridges Membrane area needed Mem. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 45 .GE Healthcare (2007) Manufacture of 100000. 10. Equipment Design Steam in place cartridges can be added Reference :. .41m D= 0.013 *1. Design of Heads: At the top: A torispherical head is used .23 mm We take thickness 6mm as this must be same as head thickness.418 bar = 0. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 46 . Crown Radius Rc = 390 mm Knuckle Radius R1 = 24 mm (6% of Rc) The thickness of the torispherical head is given as P.77 Manufacture of 100000.141 N /mm2 t = 0.141× 390 / ( 2× 140 × 0. 11.Rc. Mechanical Design of Bioreactor B003: H =0.141) + 2 = 2.85)-0. Mechanical design Of Bioreactor 11. Hence design pressure =1.J where W = stress intensification factor 1 Rc [3 ] 4 R1 = 1.2.4= 1.1. Thickness of vessel required Max pressure P = 1.W th + C 2.013 bar during cell growth.f. 11.39m 11. It is connected to the shell by means of a flanged joint. Gi = 390 +10 = 400mm External gasket diameter is calculated as. thickness = 3.0013 Gi Go = 400. 11. Design of Flanges for Head and Shell Internal gasket diameter.41 mm At the bottom: A torispherical head is welded to the shell at the bottom end.5 = 5 Under atmospheric conditions bolt load due to gasket reaction is given by: Manufacture of 100000. Go Ya mp Gi Ya mp p Gasket seating stress.54mm A flat asbestos gasket of 390 mm internal diameter and 400 mm external diameter is used.12 mm . So we use 4 mm thickness 11. Ya =52.85 ( flange joint) th = 0.5 N/mm2 Gasket factor (m) =3. Gasket seating width = b = (400-390) ×0.3. Here J = 1 Applying the same formula .41+2 = 2. Mechanical design Of Bioreactor J = joint efficiency = 0. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 47 .75 Go 1. 12 mm Manufacture of 100000. Mechanical design Of Bioreactor Wm1 = b G Ya Where G = ( Gi + Go)/2 = 800. 11.C. Ya =52.G.54 mm Gasket seating stress.42x 104 N The total bolt area is calculated on the basis of the greater load Wm1 A= where f is the permissible stress in bolt (138 N/mm2) f Therefore.56 mm So we use M26 bolts ( 78 Nos ) Pitch circle diameter (P. Bolt area = d 2 * 78 4 Hence bolt diameter = 28.G 2 .56 +12 = 469. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 48 .6 x 105 N The load under operating conditions is given by Wm2 = .5 N/mm2 Wm1 = 6. A = 41.D) = Outside diameter of gasket + 2 diameter of bolt + 12mm = 400+ 2*28.m.P 4 = 8.826 mm2 Let us use bolts of area =500 mm2 Hence number of bolts required =78.(2b).P . 6 x 105 N hg = Radial distance from gasket load reaction to basket circle = (PCD . Mechanical design Of Bioreactor Flange thickness is given by P tf G Kf 1 where K 1.69 tf = 44.3 HG Wm = total bolt load = 6.5 mm H = Total hydrostatic end force = G2P 4 = 0. 11.G)/2 = 34. 50 mm nozzle for pressure indicator 50 mm nozzle for pH indicator Manufacture of 100000. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 49 . 11.4.6 x 106 N K = 2. Design of Nozzles: The nozzles provided are as follows: 1) On shallow dished head at top: 200 mm nozzle for substrate inlet.6 mm Use 50 mm thickness of flange.5Wm h g 0. 11.165 mm Actual thickness taken =6 mm. 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.154*300/(2*1*140 . 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 50 .0.P =0.154) = 0.The area for which compensation is required is A = d ts ` = 300 x 1. Mechanical design Of Bioreactor 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. P di tn = 2fJ . 2) On torispherical head. 150 mm nozzle for sight glass.42 = 426 mm2 Manufacture of 100000. 165 mm Ho = 2.175 mm2 As + An =1181. 11.42 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 . 11. Mechanical design Of Bioreactor 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 = 1.5. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 51 .Bracket support Manufacture of 100000.From similar calculations.175 mm2 > A So reinforcement ring is need not be provided.5 x tn An = 29. it is found that no reinforcement ring is required for any of the nozzles. Design of supports Selection of support: Vertical vessels : Bracket or Skirt support H/D: 2 to 5 .tn`) tn = Actual thickness of nozzle used = 6 mm tn` = Nozzle thickness required theoretically =0. 8 /4 =33. we use Bracket support No. fh = permissible bending stress = 155 N/mm2 l= 75 mm b=h=200 mm L= 300 mm Hence . of brackets for D< 3000mm = 4.810 N Wmax L b 4 f h 0. i) Thickness of stiffener ( horizontal plate) Wmax = Σ W / no. Mechanical design Of Bioreactor H/D>5 – Skirt support Hence.7 4 b * l th b L 4 Where.51mm th = 2 mm ii) Thickness of Gusset plate 3Wmax l tg f h h 2COS Manufacture of 100000.5*1200)*9. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 52 . th = 0. of brackets (neglecting wind loads as vessel is indoors and is not very tall) = (11. 11. 6.63cm3 d = diameter of shaft Manufacture of 100000. Mechanical design Of Bioreactor Θ = 45 o Hence tg = 1. = 229.6 kWatt The continuous average torque on the shaft is given by. Tc= power / (2xxN).18 N-m The shaft must be capable of resisting 1 ½ times the continuous average torque Tm = 1.22 mm We take tg = 2 mm 11. 11. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 53 .5 x Tc = 344 N-m = 34400 N –cm Tm Zp = fs fs = Maximum permissible shear stress on shaft ( 9457 N / cm2) Zp = Polar modulus of section of the shaft d3 Zp = 16 (×d3)/16= 3. Design of shaft : Diameter of agitator = 700 mm Agitator Speed = 151 rpm Power = 3. Mechanical design Of Bioreactor d = 2. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 54 . we use a shaft of 6 cm diameter.65cm Hence. Manufacture of 100000. 11. 1 Table 12.025 m3.05m3 Diameter 0.1. Bioreactor B001 To ensure 5% by volume inoculum at every stage of fermentation. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 55 .025m3 Diameter 0.2:Sizing of B002 Type Jacketed stirred tank vessel Operating temperature 370C Operating pressure 1 atm Volume 0.3m MOC SS316 12.Seed Bioreactor B002 The sizing is done as described in Section 11. 12.1 :Sizing of B001 Type Jacketed stirred tank vessel Operating temperature 370C Operating pressure 1 atm Volume 0.2.3m Height 0.4 m MOC SS316 Manufacture of 100000. the size of B001 is taken as 0. D:Diameter of tank h:Height of tank Table 11. Equipment sizing 12. Equipment Sizing 12.4 m Height 0. 25m3/hr(Perry’s).5× Pa s Area required for centrifugation= Manufacture of 100000.Production Bioreactor B003 The sizing is done as described in section 11.5m MOC SS316 12.075 m3 Diameter 0. Equipment Sizing 12. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 56 .5m Height 0. Let the flowrate through the centrifuge be 0. 12.3 : Sizing of B003 Type Jacketed stirred tank vessel Operating temperature 370C Operating pressure 1atm Volume 0.4.07 m3.Centrifuge C001 The quantity of supernatant to be processed is 0. 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).Time taken for complete centrifugation= . :diameter of cell particle(m):2µm :density difference between cell particle and water:600kg/m3 :Vicosity of supernatant(Pas):8.1 Table 12.3. 6mm ID × 10cm Eluent A] 20mmol/L MES buffer. 12.5m No. pH 6.5.5g/L Manufacture of 100000. 4. 15min (30%B). Sizing of C001 Type Disc stack centrifuge Operating temperature 30ºC Operating pressure 1 atm G 4300 Area 53.0 Gradient 0min (10%B).41 m2 Diameter 0. linear Flow rate 1.0 B]0.0mL/min Temperature 250C Injection volume 20μL Samples A: human antibody. IgG1 B: human antibody.of discs 12 MOC SS316 12. 4. IgG1 Sample concentration 0. Protein A CHROM COLUMN F001 Type Protein A CHROM COLUMN Column Agarose resin. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 57 . Equipment Sizing Table 12.5mol/L NaCl in 20mmol/L MES buffer. pH 6. 6. 6 M guanidine hydrochloride Ligand Quaternary amine Ion Exchanger Type Strong anion exchanger Ionic Capacity 0. 7. 20% Ethanol Chemical Stability All commonly used buffers.23-0. 2-14 in-Place (CIP) BioProcess Medium Yes Average Particle Size 200 µm Binding Capacity/ml > 110 mg BSA/m medium Chromatography Medium pH Stability Working 2-12 Range Particle Size 100 µm-300 µm Manufacture of 100000. 1 M NaOH.1 Anion Exchange column STREAMLINE Q XL. ION EXCHANGE COLUMN F002 12. 12.6. Equipment Sizing 12.5 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. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 58 . nonionic detergents.33 mmol Cl-/ml medium pH Stability Cleaning. 2 M Sodium Acetate Ionic Capacity 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 > 140 mg lysozyme/ml medium Chromatography Medium Ligand Sulphopropyl pH Stability Cleaning. nonionic detergents. Cation Exchange column STREAMLINE SP XL Chemical Stability All commonly used buffers. 12. 3-14 in-Place (CIP) pH Stability Working 4-13 Range Certificate of Analysis Yes BioProcess Medium Yes Manufacture of 100000.6. 1 M NaOH.18-0.2. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 59 . 6 M guanidine hydrochloride Flow Velocity <500 cm/h Storage Conditions 4 to 30°C. 20% Ethanol + 0. Equipment Sizing 12. Virus retentive filtration F003 The product concentration in the system =11.Sizing of Filtration F003 Type Tubular membrane unit Operating temperature 30ºC Transmembrane pressure 72.5 wt %(24.An optimum flux of 0. SS304 cover Manufacture of 100000.7.00025 m/s is obtained at a trans membrane pressure of 72. Table 12.Let process take 4 hrs.64m2 No of modules 16 MOC Regenerated cellulose membrane .4 kPa Area 1.4 kPa Area 6.6. SS304 cover 12.Sizing of Filtration P004 Type Tubular membrane unit Operating temperature 30ºC Trans membrane pressure 72.4kPa. An optimum flux of 0. 12.It is concentrated 5 times.56m2 No of modules 58 MOC Polysulfone membrane . 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 60 .It is concentrated 5 times.73 g/L.000025m/s is obtained at a trans membrane pressure of 72. Table12.4kPa. Diafiltration Unit F004 The protein concentration in the system 2.8.5 . Equipment Sizing 12.62 kg/m3). 724 0.096 Centrifugal P002 3.7. Air Compressor S0001 Compressed air is required for fermentation.724 0. Sizing of S0001 Type Single stage axial flow compressor Power 4HP Capacity 0. Air flowrate:70.0016 Centrifugal P007 0.9.138 1.8.00885 Centrifugal P005 0.Pumps.02 m3/s Suction pressure 1 bar Discharge pressure 2bar MOC SS304 12.115 Centrifugal P004 0.6 1 0.24 Centrifugal P003 3. 12.724 0. They are as follows Table 12. Different pumps are used at various stages in the process. Sizing of pumps Function Capacity(m3/hr) Discharge Power(HP) Type pressure(bar) P001 2. Centrifugal Manufacture of 100000.5 bar Table 12.92m3/hr Air inlet pressure:1.025 1.09 1 0. Equipment Sizing 12.10.25 2 0. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 61 . Each pump is attached with a storage vessel for liquid.00483 1. Manufacture of 100000. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 62 . PI controller It is a proportional plus reset controller. It gives considerable offset but is simple and cheap and fast. It anticipates what the error will be in future and applies a control action proportional to the rate of change of error. Such a controller can eliminate small errors.3. it is important to maintain certain parameters at a specific value for the optimum growth of cells.1 P controller The proportional controller actuates the output proportional to the error. Different types of controllers 13. 13. However for response with constant non-zero error it gives no control action. The integral action causes the controller output to change as long as there is error in the output. Instrumentation and Process control 13.1. 13. 13. It produces zero offset but response in oscillating and sluggish 13. 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. In a bio-fermentation process. PID controller It is a proportional plus reset plus rate controller.1. The control hardware is either analog or digital.2.1. instrumentation and control plays an important role to help achieve the desired degree of productivity. The basis control strategy involves a sensor which measures the process variable and converts it into appropriate signal via the transmitter . Also for small errors it may unnecessarily bring about large control actions.1.The transmitter then sends the signal to an indicator and controller to which set point is fed for the controlled variable. Instrumentation and process control In any process. The analog system may be pneumatically operated using instrument air or electrically through wire. flow rate of cooling water in the jacket Controlled variable: Temperature of Bioreactor Manipulated variable: Flow rate of cooling water 13.2.4 bar during the sterilization. Inlet air flow rate 13.2. Although the pressure may go up to 1.PID controller is used. During the fermentation process an internal pressure of about 1. jacketed cooling water flow is used to maintain the temperature of the fermentor at 37°C.2. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 63 . Control strategy for the Bioreactor 13. Measured variable: Temperature of Bioreactor. 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 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. Pressure Control The control of pressure of the system is important from the safety point of view and to maintain sterile conditions. Instrumentation and Process control 13. Here a PI controller is used. pH Control The pH of the system needs to be maintained at about 6-7.3-1.1. Temperature Control Fermentation being an exothermic process.2. This is done using a cascade control loop with the aid of TIC01(primary controller) and FIC02(secondary controller ).2.2 bars is desired to maintain sterile conditions. As the fermentation proceeds the pH of the broth continues to increase. Manufacture of 100000.PID controller is used since no offset can be tolerated.3. Measured variable: Pressure inside Bioreactor Controlled variable: Pressure inside Bioreactor Manipulated variable: Vent air flow rate. 13. with the foam acting as the electrolyte and the vessel as earth. Manufacture of 100000. Here again a feedback control strategy is used wherein signals of level indicator are used to control the antifoamer flow rate. Instrumentation and Process control Measured variable: pH inside Bioreactor Controlled variable: pH inside Bioreactor Manipulated variable: Acid Flow rate 13. Measured variable: level inside Bioreactor Controlled variable: level inside Bioreactor Manipulated variable: Flow rate of antifoamer. 13. This is controlled by the addition of anti-foam when the liquid level (foam level) reaches a predetermined level.4. When the foam reaches the probe tip. A timer is also provided to ensure enough time for the antifoam to mix properly before more anti- foam is added. Control of foam formation There may be a rise in the liquid level due to foam formation. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 64 . A PI controller is used. a current is passed through the circuit of the probe which gets completed.2. Instrumentation and Process control TT: Temperature transmitter FT: Flow transmitter LT: Level transmitter PT: Pressure transmitter PIC: Pressure indicator and controller TIC: Temperature indicator and controller Manufacture of 100000. 13. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 65 . Guidewords: The different guidewords used and their significance is as follows: Table14. 14. Actions: Immediate activity to be performed to bring back normal functioning. High. Possible consequences: Probable repercussions of increase/decrease of parameter. Safeguards: What should be done to prevent operating problems in the future . HAZOP Studies 14. Too High Quantitative increase Applies to quantities such as flow rate and to activities such as heating Low. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 66 . Manufacture of 100000. The various terminologies used in HAZOP analysis are: Parameter: The important controlling factor.1. Guide words Guide Words Meaning Comments None The complete negation No part of the design intention is of the intention achieved. 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. 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. 14. Failure of temperature alarm controller. Failure of Increase in 1. Install thermocouple.Not decrease flow rate extra valve rate 2. temperature . 2. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 67 . Failure of temperature alarm controller. HAZOP Studies The table shown below presents HAZOP study done for the Bioreactor (B003): Table 14. Cooling Too high Failure of Increase in Increase flow rate 1. Install Alarms Too low Setting error in Decrease in Decrease flow 1. microbes cooling water 2. Manually 1. Failure of optimum for 2. Install water flow thermocouple. Increase (Bypass) control valve. Manually 1. Install 3. temperature . Install refrigeration temperature-Not rate cascade system optimum for control microbes 2. Failure of Yield 2. Too low 1.poor cascade temperature system Yield control 2.poor increase flow rate extra valve 2. HAZOP of B003 Parameter Guidewords Possible causes Possible Actions Safeguards consequences Cooling Too high 1. Reduce (Bypass) control valve. Install water refrigeration temperature. Failure of Decrease in 1. cooling water 2. Install Alarms Manufacture of 100000. Install 3. Too low Operator or Poor distribution of Manually Install alarm mechanical air. Regularly carry out maintenance of valve. Install an leakage in valve extra valve 2. Shut-off air 2. Install controller flow. HAZOP Studies Impeller Too high Operator or High Shear.Failure of Explosion 1. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 68 . Failure of manually alarm pressure 2. 14. 2. generator impeller microbes. 1. Too Low Vent open / Contamination Seal valve. Manually reduce Install alarm Speed mechanical destruction of impeller speed error microbes. Open pressure 1. increase impeller error speed None Power failure or No distribution of air Switch on backup 1. disc valve that automaticall y releases pressure. Install alarm Internal Too high 1. Install failure of and no suspension of power supply. Install a pressure pressure valve relief valve critical 2. Manufacture of 100000. preparation etc) TOTAL 65.6 Hold time 0. Using the unsteady state equations.75(considered for lag phase.755 B003 Heating 0. Batch Scheduling Activity Time(hrs) Initial Down time for 5 Transformation/ Preparation B001 Fermentation 48 Down time 2 TOTAL 50 Activity Time(hrs) BOO2 (50L) Fermentation 60 Down time 5.Bioreactor is carried out in fed. Cool down time =10 min .Batch Scheduling Proper batch scheduling is important for achieving optimal productivity.Batch Scheduling 15.75 Manufacture of 100000. Hold time = 45 min.1. 15.the time required for various activities is found as follows For sterilization of Bioreactor B002 Heat up time =36 min. Table 15. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 69 .batch mode. 69 Emptying 1.Thereafter all the batches take 282.76 days).66 Centrifuge C001 1 F001 Affinity 0. 15.5 Chromatography F003 VRF 4 F004 UF 4 F005 DF 4 FC001 Formulation 1 column The total time taken for the first batch is 282.1666 Fermentation 143.Batch Scheduling Cooling 0.45 TOTAL 146.5 Chromatography F002 Ion exchange 0.41hrs.41hrs Manufacture of 100000.(11. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 70 . To improve corrosion resistance the chromium content in the material needs to be above 12 %. which are installed inside the bioreactor vessel. 16. hardness .5.).1. Hence an optimal choice should be made which justifies the trade off between the cost and quality of material chosen. There are high requirements for the reactor vessel materials to prevent the inhibition of the microorganism growth. etc. highly aseptic environment is required. The relation between the height H and diameter D of the bioreactor is within 1. pipes. Mechanical properties This includes parameters like tensile strength. Few important characteristics to be considered when selecting a material of construction are: 16.5-2. Manufacture of 100000. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 71 . stiffness elastic modulus (Young’s modulus). For any biological process. toughness . creep resistance. The selected MOC needs to have good mechanical properties under the process conditions. biochemical. and textile industries. metal or their combination. MOC Selection For efficient working of the process. the stability of equipment is very important.MOC Selection 16. Contamination. to avoid contamination. 16. 2. demand a material with high corrosion resistance. The bioreactor vessel is made of steel. the surface finish of the material is as important as the choice of material.3.4. 16. The same applies also to any other part (sensors. Hence the material chosen should possess superior mechanical properties. The presence of aeration coupled with agitation and tendency of reduction in pH during the process. fatigue resistance. This stability to a large extent depends on its material of construction. Corrosion resistance This is an important characteristic to be considered while choosing an MOC for the considered process. pharmaceutical. 16. In industries such as the food. During sterilization all the equipments are exposed to intense temperatures and pressure. Cost The material cost plays an important role in project cost evaluation. 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 inert gas should be argon. The MOC selected for all other equipments in the plant is stainless steel. which fully replaces the air. 16. the application of the welding technology not corresponding to the requirements can cause the corrosion of the welds. With time. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 72 .MOC Selection Based on the above criteria. All the metal parts should be made from stainless steel. Welding should be carried out in a fully inert gas medium. The inner surface of the stainless steel bioreactor should be polished to about a mirror surface quality to facilitate the washing and sterilization process. Manufacture of 100000. Plant Layout: The general manufacturing site layout is as shown below: Green Belt ETP Water ETP Chilling Plant Reserved Space Steam For future growth Generation Storage Tanks Workshop And Maintenance MAIN PLANT AREA 10 Medical Control Facilities Room Store Room Records Fire Station Rest Room Keeping Administration Canteen Time Keeping Building Office Securit y Office Securit Green Belt y Parking Office Manufacture of 100000. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 73 . Plant Layout 17. 17. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 74 . 17. Plant Layout The equipment layout in the plant is as follows: S001 S002 S003 S004 B001 B002 B003 C001 S005 S0001 S006 F004 F003 F002 F001 FC001 V001 PRODUCT STORAGE Manufacture of 100000. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 75 . Project cost estimation. to take into consideration the time face the CE plant cost index needs to be taken. the following power – law model is used to deduce the cost of the equipment. The table below displays the key economic evaluation figures for the case of Rituximab. Table 18.01Costs of various materials Sr. 18. Also. (including fabrication) 1./kg) No. In cases where all data pertaining to the equipment isn’t available.63: For vessels. storage units etc. we first estimate the Cost of equipment (Delivered). SS-316L 550 Manufacture of 100000.1. 18. n= 0.Financial Analysis 18.68: For general equipment. 0.1.1. which are obtained from formulae. Carbon Steel 150 3. Material Cost (Rs. etc. 0. graphs. 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. cost – capacity correlations. For estimating the Project Cost (Fixed Capital Investment).63: For Fluid handling equipment. Financial Analysis 18. 11: Gross cost of installment Equipment Installmnent Cost (Rs) 2. 18.11 Gross Cost of installed equipment Table 18.2 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 Manufacture of 100000.1.Financial Analysis 18. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 76 . 5898 Lakhs.35898 Octrai 3 8.30706 Subtotal B 531.207694 Transportation 3 8.12: Breakup of OSBL % of Equipment Cost Component cost (lakhs) Equipment 273. instrumentation.Financial Analysis The gross cost of installed equipment = Rs.07694 Total A 506. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 77 .03847 GRAND TOTAL plant and machine( Lakh) 673.1 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.5898 Piping 20 54.2. 18. it would be reasonable to assume that the cost of the OSBL facilities would be a fraction of the Battery Limit plant. 18.03847 OSBL 30 82.03847 insulation and painting 2 5.1411 Spare 5 % 0f A 25. 18.71796 Electricity 15 41. Therefore.2 Estimation of plant and machinery cost 18.207694 Excise 15 41. In a similar way the piping.4482 Packaging and forwarding 3 8. installation and other components of plant and machinery costs are estimated. 273.71796 Instrumentation 20 54.7149 Manufacture of 100000. electrical.735898 Installation 15 41.207694 Insurance 1 2. We assume that it is 30 % the cost of battery-limit equipment.471796 VAT 10 27.2 Plant and Machinery cost Table18.2. D.2.794 Plant and machinary cost 50 673.C website Land and site development cost 134.201682623 Acres Manufacture of 100000.4229 Expenses Total Project Cost (Lac) 100 1347.743 Building and civil work 8 107.47Crores Note: Price of Land is found from G.8651 m2 Area of land 2. (Mahajani & Mokashi.794 Miscellaneous Fixed Assets (MFA) 3 40.3 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.43Lakh = Rs 13. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 78 .3715 pre operative expenses 13 175.Financial Analysis 18. Table 18.715 Khow how and engineering fees 8 107.13 Breakup of Total Capital Cost Component % Cost Land and site development 10 134. 2005).7429765 Lakh GIDC Rate 1500 Rs/m2 Area of land 8982. 18.166 Preliminary & Capital issue related 3 40.I.43 Total Project Cost = Rs 1347.4229 Contingency 5 67. A provision is made for maintenance spares as 0. The total plant and machinery cost of the project = 673.7149 lakhs. Thus. 6.2 Product and Utility in stock The amount is estimated at the cost of production. 20.3 Maintenance Spares Calculating the plant and machinery item of project cost for 1 month.4 Other fixed costs These are estimated as 10% of the product inventory cost.1% of the plant and machinery item of the project cost for one month. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 79 . Thus. therefore. 20. 0.31578947 lakhs. of Total Product days Amount(kg) Cost(Rs)/Kg Cost(Rs) Product 15 0. 18. maintenance spare inventory cost = Rs. 20. other fixed expenses = 10% of (644) lakhs.Financial Analysis 20.4737 TOTAL PRODUCT COST ( Rs) 6315789.1 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 estimates are based on the guideline by Mahajani & Mokashi (2005).1 Product Streams as WIP and Utility costs for 1 batch No. The inventory. = Rs. Manufacture of 100000. Estimation of Working Capital Working capital is the capital required to make the project work perform.474 20. The following are the components of working capital.67374883 lakhs. Table 20. is not applicable for raw materials.5 12000000 6000000 0 Utilities 15 5 % of total 315789. 31578947 TWC (Lac) 119.3802571 Manufacture of 100000.232858 Product inventory cost 63.2 Total working capital break-up Parameter Cost(lakh) Raw material inventory cost 49.Financial Analysis Table 20. 18.5 Working capital source Following table shows source of total working capital.67371488 Other fixed cost 6.84506427 Total Working Capital ( Lac) 119.380257 20.3 Source of working capital Component Contribution Cost in lakh Borrowed WC 75 % TWC 89.1578947 Thus. maintenance spare inventory cost 0. Table 20. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 80 .53519 Margin money 25% TWC 29. ) 576750 Manufacture of 100000. 15/m3 750 Electricity 58000 KW. Total Cost of Production 19.2: Cost of Utilities Annual Utilities requirement requirement Rate (Rs.25 2500 5 12500 Total raw material cost 98465716 19. Media Sol 129 368 2580 949440 serum free media 246 18000 4920 88560000 Air 10525 0 210500 0 H3PO4 6275 8.4/kg 112000 Total Utilities cost per annum (Rs.2 231240 2127408 ION eq buff 1730 11. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 79 ./ unit) Cost per annum Cooling water 50000 Kg Rs. 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. A reg buff 3174 10 63480 634800 Pro. This has significant impact on selling price and hence the profitability of the project.5 125500 1066750 NaOH 5892 15 117840 1767600 Pro.8 /KW.1.A elution 5317 9.1 Raw Material Cost Table 19.2 106340 978328 Pro A equil 11562 9. Following are the main components of cost of production.hr Rs.1: Cost of Raw Materials Raw material Batch req Rate(Rs/kg) Annual Cost (Rs) (kg) req(kg) Inoc. Sulfate 74 480 1480 710400 polysorbate80 2 110 40 4400 sodium citrt dihydrate 0.5 1940 35890 Nacl(1M) 1065 22 21300 468600 amm.2 Utilities Cost Table 19. 19.2 34600 387520 ION wash buff 1732 22 34640 762080 IOX el buff 97 18.hr 464000 Steam 80000 kg Rs. It is assumed that the plant runs continuously for 300 days in a year. 3 Indirect cost Table 19:3 Indirect Product Cost Component Contribution Total Cost (Rs.5 Plant Overheads Plant overheads = 40 % (OLC + Supervision + Repairs and Maintenance Cost) = Rs. In lakh) 67.930 lakh Manufacture of 100000.47429765 Total fixed charges ( Rs.37148825 19. Total Cost of Production Thus.8971906 1 % of project Insurance cost 13. 19. In lakh) 252.930 lakh Total plant Overheads (Rs. In Component Contribution lakhs) 4 % of project Local tax cost 53. 88.37148825 Supervision 15 % of OLC 20. Variable Cost = Raw Material Cost + Utility Cost = 990.6430809 19. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 80 . In lakh) = 88.21144648 Labour 15 % of OLC 20. In lakhs) Operating labour cost (OLC) 10 % of project cost 134.7429765 Repairs and maintainance 5 % of project cost 67.42466 lakhs 19.4 Fixed Charges Table 19:4 Fixed Charges Total Cost (Rs.21144648 Supplier 15 % of M & R 10.10572324 Indirect product cost (Rs. 840592 in lakh.415 lakh Fixed cost = Rs. Now.69485953 cost Generl expences ( Rs. 19. Cost of production = Rs. 446.415 lakh. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 81 .68574413 Distribution and selling 0. In lakh) 36.92=14370000/kg 19.09 x (990. 446. In laks 1.8 Cost of Production Cost of production = Variable cost + Fixed cost Total cost of production =Rs1436. 19.9 Comments on Economic Feasibility Following is the production capacity of products and revenue generated from selling product: total Products Rate ($/kg) Annual Capacity(Kg) Rate(Rs)/Kg Cost(Rs) Product 300 10 18000000 180000000 Manufacture of 100000. Total Cost of Production 19. We manufacture 10 kg of RITUXIMAB.6 General Expenses Table 19:5 General Expenses Component Contribution Total Cost (Rs.0903/- Therefore fixed cost = Total indirect cost + Fixed charges + Plant Overheads + General Expenses + Salary and Wages = Rs.7 Salary & Wages Salary and Wages = 9 % of variable cost = 0.2 % of project 2.42) Salary and Wages = Rs. In lakhs) Administration cost 25 % of OLC 33. 14368405.38060366 19. 37 5 Plant overheads 88.65 2 Utilities 5.42 3 Fixed Indirect cost 252.38 7 Salary and wages 10.90 Fixed cost of Production 446. Since the project is economically feasible.10 Summary Table 19. in Lakh) 1 Variable Raw material 984.76 Variable Cost of Production 990. No.00 Manufacture of 100000. We can proceed to estimating the working capital and do further financial analysis of the project. 3630000/kg and therefore Rs36300000 lakh / annum.64 4 Total fixed charges 67. Total Cost of Production Weighted average selling price of product = Rs.6 Summary of Production Cost Sr.40 Total cost of Production 1437. 19. 19. 1800000/kg Hence there is an earning of Rs.93 6 General expenses 36. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 82 . Type Component Cost( Rs. 2 Product and Utility in stock The amount is estimated at the cost of production. = Rs. Thus. A provision is made for maintenance spares as 0. Thus. 0. 6. The total plant and machinery cost of the project = 673. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 83 . other fixed expenses = 10% of (644) lakhs. 20. The following are the components of working capital.1 Product Streams as WIP and Utility costs for 1 batch No. Estimation of Working Capital Working capital is the capital required to make the project work perform. Manufacture of 100000.5 12000000 6000000 0 Utilities 15 5 % of total 315789. Steam is also generated in plant from waste heat boiler and air is compressed whenever required so no need to store it.1% of the plant and machinery item of the project cost for one month. The inventory.31578947 lakhs. Table 20. Estimation of Working Capital 20.474 20.7149 lakhs. 20. therefore. maintenance spare inventory cost = Rs. of Total Product days Amount(kg) Cost(Rs)/Kg Cost(Rs) Product 15 0. 20.4 Other fixed costs These are estimated as 10% of the product inventory cost.3 Maintenance Spares Calculating the plant and machinery item of project cost for 1 month.67374883 lakhs. 20.1 Raw Materials We generally keep an inventory of 1 batch as storage of Raw Materials as work in progress. is not applicable for raw materials.4737 TOTAL PRODUCT COST ( Rs) 6315789. The estimates are based on the guideline by Mahajani & Mokashi (2005). 67371488 Other fixed cost 6. Table 20.31578947 TWC (Lac) 119.5 Working capital source Following table shows source of total working capital.232858 Product inventory cost 63. 20.2 Total working capital break-up Parameter Cost(lakh) Raw material inventory cost 49.380257 20.3802571 Manufacture of 100000.3 Source of working capital Component Contribution Cost in lakh Borrowed WC 75 % TWC 89.53519 Margin money 25% TWC 29. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 84 . Estimation of Working Capital Table 20.1578947 Thus.84506427 Total Working Capital ( Lac) 119. maintenance spare inventory cost 0. 1 Estimation of Financial Expenses Term loan repayment period = 10 years.2504 lac 34% Borrowed working capital 89. Financial Analysis Financial Analysis involves estimation of financial expenses.9719 lac Promotors contribution 274.5:1 Term Loan 808. Table 21. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 85 . 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. depreciation values. annually in equal half annual installments Interest is found by computing simple interest for that period.1 Project Financing Components Total fixed capital investment 1347. 21. Manufacture of 100000.8757 lac 51% Organizational participation 80.43 Lac Debt : Equity 1. Estimation of Financial Expenses 21.84579 lac 15% Public issue 183.53519 lac is @ 16 % installment for 4 years 21. after which equal installments are paid Working Capital Repayment Period = 4 year.4579 lac is @12 % pa Equity participation 538. yearly in equal half annual installments Moratorium period = 2 years. 67 19 146.13 32.78 13 496.15 14.03 5.51 80.58 26.79 53.43 15.98 16 336. Estimation of Financial Expenses Table 21. 21.51 0.78 2.00 743.76 80.81 50.80 71.20 42.36 15.00 708.51 808.49 80.00 146.98 20.46 48.46 2 808.45 15.76 9.42 15.22 13.84 18 213.00 277.20 8 708.17 80.47 4.33 44.58 14 446.49 37.58 51.53 75.38 80.60 5 51.15 6 40.46 4 808.60 15.13 11.35 80.46 48. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 86 .98 10.82 15.16 8.58 40.00 Table 21.70 40.62 33.37 80.51 0.03 3 72.00 776.94 37.58 14.51 808.86 42.20 17 277.24 39.09 15.47 20 75.21 12.59 7 743.00 336.60 56.38 23.00 630.70 9 670.65 47.37 15.47 80.00 588.58 81.49 9.51 0.20 80.63 63.94 10 630.97 6 776.21 4.62 35.83 67.2 Term Loan Scheduling Principle Principle Installment Principle at Installment remaining Interest payable paid paid term end Rs in Lac Rs in Lac Rs in Lac Rs in Lac Rs in Lac 1 808.00 75.46 5 808.00 496.97 46.80 35.14 80.67 80.13 12 544.00 213.67 8.00 48.00 544.78 7 27.78 80.00 48.00 48.46 48.12 2 81.51 808.15 3.40 80.51 0.21 4 62.46 3 808.38 80.00 0.20 16.78 29.58 62.00 393.38 15 393.22 59.51 31.46 48.00 48.43 1.80 11 588.20 80.59 44.51 808.58 0.46 48.43 8 14.19 80.00 Manufacture of 100000.60 4.00 446.00 670.12 6.58 27.58 72.84 12.54 7.3 Borrowed Working Capital Repayment Schedule Principle Principle Installment Principle at Installment remaining Intrest payble paid paid year end Rs in Lac Rs in Lac Rs in Lac Rs in Lac Rs in Lac 1 89. WDV: Written Down Value Method. Manufacture of 100000.42 5. %. DWDV = Depreciation rate by written down value method. In case of straight line method. the value of asset at the end of any year n is given by: Va Vo 1 DWDV n Equation 23.1 In case of written down value method (also known as accelerated depreciation method). 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 87 . Vo = original value.71 10.fixed assets (MFA) 40. %.00 SLM: Straight line method.2 In the above equations: Va = Value of asset at the end of year n. The rates of depreciation are fixed by the Income Tax Act and can be revised during the budget.2 Depreciation Schedule To estimate depreciation.00 Misc.4 Depreciation Rates Depreciation rate Cost Component Value Depreciation rate Rs in Lac SLM (%) WDV (%) Land and site development ( L&D) 134. DSLM = Depreciation rate by straight line method. 21. 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 23.34 10.00 Plant and Machinery (P & M) 673.74 Building and Construction ( B& C) 107. in their corresponding proportions. Table 21.34 25. Following table contains the depreciation rates are used for the estimation of the working results.79 3. the preoperative expenses and contingencies are distributed among the other project cost components. Estimation of Financial Expenses 21.00 20. 82 8 2.86 75.47 3.02 5.16 69.36 3.3 Manufacture of 100000.28 142.73 69.6 6.06 75.5 Depreciation Schedule Year MFA B&C P&M Total SLM WDV SLM WDV SLM WDV SLM WDV 1 2.86 10 2.02 3.09 3.32 75.30 2 2.4 Break-Even Analysis The following assumptions are made while carrying out the break-even analysis (Mahajani & Mokashi.67 6 2.86 69.78 69.66 126.65 75.66 16. Estimation of Financial Expenses The depreciation schedule is as follows: Table 21.66 168.02 6.64 69.29 75.28 83.66 39.02 1.14 3.33 9 2.70 3.65 4 2.02 1.66 29.05 5 2. The equation used to estimate the break even capacity is: S * X= V * X + F Equation 23.73 69.02 4.66 71.28 37.31 3.6 5.6 8. depreciation.07 69.66 53.65 3.6 4. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 88 .18 69. Whatever is produced is sold immediately.36 69. The market conditions are ideal.6 7.6 5.3 Sales Realisation Table 21. interest.28 48.28 29.17 3.66 94.98 7 2.6 7.98 75.43 75. The following annualized (fixed) costs are not dependant on capacity utilization.70 69. The selling expenses are fixed as some % of sales.12 3. charges on term loan.97 75.60 10.02 2.91 21.6 4.02 1.28 17.6 Sales Realisation Product Annual Selling Price ( Sales( production Rs/kg) Lac/Year) Rituximab 10 18000000 1800 21.49 3 2.28 187.09 3. 21.74 75.66 22.02 8. namely.66 12.48 75. 2005): All expenses are bifurcated into fixed and variable costs.28 108.6 9.02 2.28 22.28 63. F = 326.08 lakh/ annum V= Similarly.004027ton/ annum X =40. Estimation of Financial Expenses where. Manufacture of 100000. 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. the sale price for products in the above proportion can be calculated as: S= n X= 0. 21. X = F/(S-V) Equation 23. administrative expenses.28 %. Substituting the required values. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 89 .4 The fixed cost of production includes the overheads. average financial expenses and depreciation (SLM). Total capital investment 130222198. Profit margin = ( Gross profit / Sales) * 100 Equation 21. ROI is given by. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 90 .5 Ratio Analysis 21.1 Rs. Estimation of Financial Expenses 21. Gross profit 36300000 Rs. Return on Investment (ROI): Assuming 100% capacity utilization.16 % D.33 Manufacture of 100000. Payback Period Assuming 100 % capacity utilization Table 21.7 Payback Period Gross cost of production 143700000 Rs. 21. Gross Profit * 100 Equation 21.5.5 Total Capital Investment Return on Investment = 27. Profit Margin: Assuming 100% capacity utilization.7 Net assets turnover = 1.78% % C.587388378 Years Payback period 3 Year and 7months B. Net Assets Turnover: Net assets turnover = (Sales / Project cost) Equation 21.6 Profit margin = 20.87 65. annual sales 180000000 Rs. 3.1 Performance Ratios A. 5:1 B.5. A dividend of 20% is assumed from the first year. Estimation of Financial Expenses 21.6. 21. Interest Cover = Gross profit / Interest Equation 21.26 21. Estimates of Working Results The projected working results for 10 years are presented in tabulated form. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 91 .2 Financial Ratios A. 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) Manufacture of 100000. D:E Ratio Debt : Equity = 1. Interest Cover: 100% capacity utilization is assumed and interest cover is calculated for the first year.8 Interest Cover = 3. 3874 23.0000 4.4625 13.2836 75.0080 0.9073 Operating profit 183.0000 Capacity utilisation 90.0721 55.0501 174.4000 290.0000 95. 21.9 Estimate of working capital for years 6-10 (All figures in Rs.8 Estimate of working capital for years 1-5(All figures in Rs.0000 Gross cost of production 1005.2539 274.1273 81.9909 128.5496 94.2836 Depriciation ( WDV) 120.9271 Corporate profit 11.5557 Table 21.2836 75.0149 97.3337 250.0090 Sales 1260.1478 13.2206 193.6950 310.7643 Corporate profit 62.0000 100.0000 9.1073 78.8748 130.8500 344.3000 1365.9000 1149.9226 75.6000 1149.3237 215.2100 115.0000 % capacity 0.0000 Gross cost of production 1293.0090 0.0000 Financial expenses 67.0091 173.8020 213.0401 139.9825 37.1000 290.1706 Manufacture of 100000.6000 1293.5184 137.1500 1365.4853 148.8500 363.1238 Profit for devident 70.4245 271.5085 93.2857 167.3880 Taxable profit 209.2362 165.0523 63.9000 1005.0000 Gross profit 326.0080 0.5293 Profit after tax 195.0000 5.1500 1437.6412 150.5197 240. (Lakhs).5680 Profit after devidend 56.0000 8.8751 42.1255 87.0000 80.3576 75.0021 91.8004 18.0981 Depriciation ( SLM) 75.0100 0.1969 94.0149 95.8394 18.6725 207.0000 90.2017 73.2404 184.5537 190.1424 Profit for devident 120.3000 Gross profit 254.6015 43.2836 Depriciation ( WDV) 48.8588 Dividend 24.0000 80.6045 25.8015 119.4147 110.2038 99.4781 Profit after tax 146.0700 108.8376 144.0000 2.0000 1710.0000 70.0000 1620.7446 258.6452 83.9370 117.1037 27.0000 10.6795 331.0100 Sales 1620.2701 115.8402 devidend 14.0000 7.3889 132.5912 55.4410 212.0000 363.0070 0.9649 225.6748 Operating profit 81.0000 95.0000 1440.8241 29.0151 86.0100 34.0000 100.3284 Depriciation ( SLM) 75.2721 Net cash accural 131.7000 Financial expenses 97.0000 % capacity 0.3333 22.0095 0.8472 33.2836 75.0625 12.1212 219.5675 28.8332 169.7389 69.8218 29.0000 1800.0000 1437.1000 254.2836 75.2836 75.0000 1710.6293 120. except capacity and utilization) Year 1.4000 326. (Lakhs).3183 Taxable profit 36.0070 0.2836 75.7000 344.7421 250.0000 3.9718 Profit after dividend 96.0000 Capacity utilisation 70. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 92 .0000 1800.6983 185.0225 144.0095 0. Estimation of Financial Expenses Table 21.0000 1440.8870 Net cash accrual 171.8015 81.0000 1260.8580 17.2836 75.9888 36.4805 169. except capacity and utilization) Year 6.9754 56.2836 75. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 93 .192 = 19. D.986746 lakhs. Estimation of Financial Expenses 21.30 (assume). We have WACC = 0.5:1 iT= 0. 21. It takes two years for the construction of project.12 rE= 0.9 DE For our project. D:E = 1.2% B. assume that Rs. PV = Rs. Present Value of Investment The total capital investment is Rs. Weighted Average Cost of Capital The weighted average cost of capital is given by: D i E r WACC T E Equation 21. Substituting the values.5% (> WACC) C. 6000 lakhs are spent in the first year of investment and the rest are spent in the second year. Pn denotes the Gross Profit Projection for the nth year. Out of these. 7493 lakhs. 1670. Hurdle Rate of Return (K): K= 22. Present Value of Profit (PV) The present value of profit is given by: ∑ Equation 21. we have.10 where. Manufacture of 100000.7 Discounted Profit Flow Analysis A. 056 Lakhs The project thus seems attractive. F.11 = 256.13 IRR = 52.2% As IRR is greater than WACC. E. project is attractive. Estimation of Financial Expenses Present Value of Investment = Io (1 K ) I1I2 = Rs. 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. Net Present value of Profit The net present value of profit is given by: ∑ Equation 21. 21.1414.18 G. Profitability Index: The Profitability Index is given by: ∑ Equation 21. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 94 . Manufacture of 100000.92975akhs.12 PI = 1. cycle time of 1 day. Utilities and effluent treatment 22. Hence both the raw materials and the products need to be stored in extremely sterile environment. In order to retain its activity for a long time it is stored at -25°C. Total cooling water required =214788+520+1042+5. Utilities and Effluent treatment 22.cooling water and electricity Amount of steam required for sterilization of Bioreactor =2280+11+5. Storage. Hence total steam requirements is 4000kg Cooling water is consumed during sterilization and the fermentation process. Utilities The utilities required in the plant are 4 bar saturated steam . Storage.001 ×∆T × Vc × (Kc/Kc-1) Water losses per batch are = 2. Cooling water is re-circulated and so for annual requirement we only need to take consideration losses of evaporation and other losses. Storage The entire process demands high level of monosepsis.73 ×0. Let. The raw materials are mostly biological grade materials and are stable at room temperature.66 TPB.5×3600×85=1900tonnes≈1900 m3. So water required per batch is 126. The product cannot be exposed to high temperature. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 95 . 22.5≈3000kg.Besides steam is also required to sterilize the pipelines and other equipment. Water losses = M = 1.2.Besides water forms a major portion on the fermentation medium too. 22.5 Ton Annual fresh water required is 50 Ton Manufacture of 100000.1. Solid Effluent treatment Around 5 kg of sludge/batch is generated. This sludge can be reused. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 96 . In order to decrease the odor intensity.or by stabilization via chemical treatment with cement. by direct land application for soil conditioning and fertilization . The basic aerobic fermentation can be represented by the following equation 22. Utilities and Effluent treatment Table19.2. 22.3. Gas effluent treatment The fermentation process causes a lot of odor problems because of various volatile organic compounds let out into the atmosphere during the process.3. Storage. Manufacture of 100000. 1.Utility requirements Utility Amount/batch Steam 4000 kg Water 1900 m3 Electricity 2900 kW.1. Effluent treatment Various solid. lime etc for road subsoil additives. sodium silicate.3. cement manufacturing etc. the vent gases are passed through biofilters before letting them out to the atmosphere .hr 22. 22. liquid and gas effluents are generated during the process. total project cost comes out to be Rs. 100% product sale and negligible losses in material and energy. This even gives an opportunity of forward integration of the plant to any of the products mentioned above. etc. Maharashtra ensures that raw material availability is never a problem as well as the export and import is easy. 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 100 . which has contributed in reduction of the payback period. Some of the assumptions made are stability of raw material.18 (greater than 1) shows the profitability of the project. This report justifies the need to conduct a detailed feasibility report to come up with more realistic figures. It has been assumed that the plant functions to its full capacity from commencement of production. Conclusions The technical and economic feasibility of putting up a 100000 vials of Rituximab plant in India has been reviewed. To get clearer scenario. a detailed feasibility report needs to be done. Manufacture of 100000. 1437 lakhs giving a return on investment (ROI) of 65. the project on the basis of the above analysis indefinitely profitable and economically feasible. On the basis of the calculations for the economic feasibility of the process. the project costs may vary depending process licensing fees. Also. Conclusions 24. The profitability index value of 1. it is to be noted that this feasibility study has been done considering some ideal situations. we can conclude that setting up this plant in India would lead to substantial profits.41 % (payback period of 43 months). Thus. product prices and market demands. So. The installed capacity of 10 kg offers great flexibility in meeting the demands of the product. The demand of the above mentioned products is ever increasing hence the market can be assumed to be stable and increasing. The time value of money also has not been accounted for. 24. shipping of proprietary equipment. The location at Raigad. However. Volume 6. In: Gottschalk U. “Perry’s Chemical Engineers Handbook”. Diekmann SAD. Penny R. Prentice Hall of India Pvt. (1990) Manufacture of 100000. Hoboken. Downstream processing of monoclonal antibodies: current practices and future opportunities. Green D... References Derouazi M. (1948).. O. Sydney. Kelley B. John Wiley and Sons Inc. Process Scale Purification of Antibodies. ed. September/October 2009. 25. (2006) Stephanopoulos G. J Chromatogr B 2007. 7th Edition. Gunhan S. Lee A.K. Single-Use Bioreactors for the Clinical Production of Monoclonal Antibodies: A Study to Analyze the Performance of a CHO Cell Line and the Quality of the Produced Monoclonal Antibody. BMC Proceedings 5.. “Chemical Process Equipment: Selection and Design”. 87. 1-23. Biotechnol Prog 2007. New Delhi... 848:29-39. 2004: 537–545. Landes Bioscience. H. mAbs 1:5. H.J. Butterworth-Heinemann (an imprint of Elsevier). Downstream processing of monoclonal antibodies-application of platform approaches.J. “Coulson and Richardson’s Chemical Engineering Series: Chemical Engineering Design”. Biotechnol. and Couper R. (1995) Uhlig. Blank G..W.. Ltd.M. Newton (USA). S. Perry R. (1999) Shukla AA. 23:995-1008 Kelley B Industrialization of mAb production technology The bioprocessing industry at a crossroads. 4th Edition. New York. Butterworth-Heinemann. “Chemical Process Control: An Introduction to Theory and Practice”. Very large scale monoclonal antibody purification: The case for conventional unit operations. 2001: 103 Kelley B. and Malony J. W.. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 101 .H. Low D. McGraw-Hill. “Corrosion Handbook”. Bioeng. Fair R. R. Serum-Free Large-Scale Transient Transfection of CHO Cells. Tressel T. 443-452..References 25. Sinott. Walas. NJ: J Wiley & Sons 2009. Hubbard B. New Delhi. S.H. Butterworth-Heinemann. New York. Great Britain.. New Delhi... 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 102 . Volume 2. Macmillan India Limited. Maclmillan India Ltd. and Mahajani. L. C.V. V. 1989.. 6th edition. (2007) Jackson A. V. The Environmental (Protection) Act. J.References Rao. J. “Environmental Pollution Control Engineering”.. (1995) Mahajani. Wiley Eastern Ltd. M. Storage and Import of Hazardous Chemical Rules... “Chemical Project Economics”. McGraw Hill Inc. (2001) Joshi. and Mokashi. 25.T. and Harriot. “Coulson and Richardson’s Chemical Engineering: Particle Technology and Separation Processes”..M. 1986 The Manufacture. (1996) Richardson.R. C.F. Government of India. V. New Delhi.”Process Engineering in Biotechnology”. Prentice Hall International Series in In the physical and chemical engineering sciences. and Harker. 3rd Edition. P. S. Smith. “Unit Operations of Chemical Engineering”. V. (2005) McCabe. Manufacture of 100000. J. with Backhurst.. W. J. 5th Edition. India. “Process Equipment Design”. 1200). and for use in treating certain types of rheumatoid arthritis. colorless liquid. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 103 . Routes of Exposure Direct contact with eyes. skin.000 Daltons) and its poor potential for absorption Section 3 – Composition/Information on Ingredients Component(s) CAS Number Rituximab 174722-31-7 Sodium Citrate 6132-04-3 Polysorbate 80 9005-65-6 Sodium Chloride 7647-14-5 Formula (drug substance): Recombinant chimeric mouse/ human monoclonal antibody to CD20 antigen. This product is a clear. or mucous membranes is the possible primary route of occupational exposure. a protein that is not well absorbed by inhalation or by contact with eyes. Manufacture of 100000. No adverse health effects through these routes are expected to occur in occupational exposure conditions due to the large size of rituximab (a full length monoclonal antibody with a molecular weight of ~145. Although the health effects of occupational exposure to this product are not fully known or characterized. It derives its biotherapeutic benefit from a monoclonal antibody (rituximab). Adverse health effects have been observed in patients following intravenous (IV) injection of therapeutic doses for treatment of non-Hodgkin's lymphoma. or mucous membranes. no adverse effects are anticipated as a result of occupational or incidental exposure. skin. Material And Safety Data Sheets Appendix A: Material Safety Data Sheet Section 1 – Product Product Name Information RITUXIMAB Section 2 – Hazardous Ingredients / Identity Information Emergency Overview Rituximab is considered hazardous per the criteria under the OSHA Hazard Communication Standard (29 CFR 1910. No special fire fighting measures. Section 7 – Handling and Storage Refrigeration (2-8°C. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 104 . use safety glasses with side shields. Material And Safety Data Sheets Section 4 – First Aid Measures Eye/Skin Contact Immediately flush eyes thoroughly with water or wash skin for at least 5 minutes as a prudent chemical hygiene practice. Dispose of collected material in accordance with applicable waste disposal regulations. Section 6 – Accidental Release Measures Take proper precaution to minimize exposure by using appropriate personal protective equipment. 36-46°F) is advised to maintain longer pharmacological activity. such as latex gloves and lab coat. Manufacture of 100000. Report exposure to supervisor. Protect from sunlight. Section 8 – Exposure Control and Personal Protective Equipment Skin Protection As a prudent chemical hygiene practice. wear protective equipment that minimizes the potential for skin contact. Eye Protection As a prudent chemical hygiene practice. Other Clean all protective equipment after use. Section 5 – Fire Fighting Measures Flammability/ Explosivity Not flammable or explosive. Avoid agitation. If material is released or spilled. soak up material with absorbent material and wash spill area thoroughly with soap and water. Wash hands and other potentially exposed areas immediately after handling material. 21 and 22. However.5 Boiling Point (degrees C): ~100 Melting Point Not applicable Vapor Pressure: Nil Solubility in Water: Soluble Evaporation Rate: Equal to water Appearance: Clear. at 15. 50 or 100 mg/kg/week. The 100 mg/kg/week dose resulted in exposures of 0. and then weekly on post-coitum days 29. Rituximab was administered as loading doses on post-coitum days 20. at 20.5 or 75 mg/kg/day. 36. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 105 .000 Daltons pH: 6.8-fold a human 2 g dose based on AUC. there was no evidence of teratogenicity under the conditions of the experiment. Material And Safety Data Sheets Section 9 – Physical and Chemical Properties Molecular Weight ~145. Animals were administered rituximab via the intravenous route during early gestation (organogenesis period. there are no adequate and well-controlled studies in pregnant women. 43 and 50. Although rituximab has been shown to cross the monkey placenta. Manufacture of 100000. colorless liquid Specific Gravity: ~1 Section 10 – Stability and Reactivity Stability: Stable Hazardous Polymerization: Will not occur Hazardous Decomposition Products: None expected Section 11 – Toxicological Information Eye No data available Skin No data available Reproductive and Developmental Toxicity An embryo-fetal developmental toxicity study was performed on pregnant cynomolgus monkeys. post-coitum days 20 through 50). 37. and severe mucocutaneous reactions Section 12 – Ecological Information Persistence and Degradability This product is protein based and will rapidly degrade in the environment. and for use in treating certain types of rheumatoid arthritis include fatal infusion reactions. Manufacture of 100000. The above information is offered in good faith and with the belief that it is accurate. state. a substitute for consultation with appropriately trained personnel. and local guidelines. Material And Safety Data Sheets Carcinogenicity and Mutagenicity No long term animal studies have been performed to establish the carcinogenic or mutagenic potential of Rituximab. Section 14 – Other Information No additional information. 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 106 . While efforts are made to provide useful information relating to handling. Medical Conditions Aggravated by Exposure None known or reported. in the event of an adverse incident associated with this product. Clinical/Human Studie Adverse health effects that have been observed in patients following intravenous (IV) injection of therapeutic doses for treatment of non-Hodgkin's lymphoma. Tumor Lysis Syndrome (TLS). and is not intended to be. this Material Safety Data Sheet (MSDS) is not. Aquatic Toxicity No data available Section 13 – Disposal Considerations Dispose of waste residues according to prescribed federal. Vibhute B. S001 B001 Seed Bioreactor1 P-1 B002 Inoculum Train P-5 S002 B003 Production Bioreactor 3 C001 Centrifuge P-2 P-3 S003 S004 B001 B002 P-7 F001 Affinity chromatography Unit P-4 P-6 F002 Ion Exchange Unit P-8 P-9 P-9 F003 Virus Retentive Filtration Unit V-6 P-10 F004 Difiltration Unit V-4 FC001 Formulation Unit B003 V001 Vials Filling Tank S005 S001 Substrate Medium Tank V-7 P-11 P-12 P-16 S002 Antifoam Tank P-15 P-13 S003 Acid TAnk S0001 E55 P001 P-18 S004 Hydroxide Tank P-19 S005 Product container P-21 P-20 E001 Fired heat exchanger S0001 Compressor P005 P004 P003 P002 C001 P-32 P-29 P-27 P-24 S006 P-31 P-28 P-25 P-23 F004 F003 P-22 F002 F001 P-33 P-30 FC001 Process Flow Diagram P-34 Design a Plant o manufacture 100000.Chem Engg 2013-2014 . 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB V001 P-35 Anil V.