Design a Plant for Rituximab Production

DESIGN A PLANT
To manufacture 100000, 10ml fill vials
per annum at 10mg/ml strength of
RITUXIMAB
HOME PAPER SUBMISSION
BACHELOR OF CHEMICAL ENGINEERING
BY
ANIL V VIBHUTE
INSTITUTE OF CHEMICAL TECHNOLOGY
MATUNGA, MUMBAI - 400019
2013-2014
YG

INDEX
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB
Sr.no Title Pg No.
1 Introduction 1
2 Executive summary 5
3. Process selection 7
4. Process Description 9
5 Kinetics and Thermodynamics of Process 17
5.1 Kinetics 17
5.2 Thermodynamics 19
6 Block Diagram 20
7 Site Selection 21
8 Material Balance 25
9 Energy balance 34
10. Equipment Design 36
11 Mechanical design of F003 46
12 Equipment Sizing 55
13 Instrumentation and Process control 62
14 Hazard and Operability(HAZOP) Analysis 66
15 Batch Scheduling 69
16 MOC Selection 71
17 Plant Layout 73
18 Financial Analysis 75
19 Total Cost of Production 79
20 Estimation of Working Capital 83
21 Estimation of Financial Expenses 85
22 Storage, Utilities and Effluent Treatment 95

INDEX
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB
23 Safety ,Health and Environment 97
24 Conclusions 100
25 References 101
26 Appendix A 103

1. Introduction
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 1
1. Introduction.
This document is a project feasibility analysis for manufacture of 100000, 10ml fill vials per
annum at 10mg/ml strength of RITUXIMAB.
1.1.Rituximab
Finding a cure for cancer has always been a goal for many health care professionals. Many have
tried, but few have made as much of a stride as Dr. Antonio Grillo-López. He, along with several
colleagues, pioneered a new drug named rituximab that serves as the first FDA approved
antibody to treat cancer. Depending on the severity of the cancer, this drug could either
completely treat or extend the lifetime of the patient. This extraordinary feat had very humble
beginnings.

1. Introduction
A Rituximab is a genetically engineered chimeric murine/human monoclonal antibody consisting
of a glycosylated IgG1 kappa immunoglobulin with murine light- and heavy-chain variable
regions (Fab domain) and human kappa and gamma-1 constant regions (Fc domain). Directed
against the CD20 antigen. Rituximab is produced by mammalian cell (Chinese Hamster Ovary)
suspension culture in a nutrient medium containing the antibiotic gentamicin. Gentamicin is not
detectable in the final product. Rituximab is a sterile, clear, colorless, preservative-free liquid
concentrate for intravenous administration. Rituximab is supplied at a concentration of 10
mg/mL in either 100 mg/10 mL or 500 mg/50 mL single-use vials. The product is formulated in
polysorbate 80 (0.7 mg/mL), sodium citrate dihydrate (7.35 mg/mL), sodium chloride (9 mg/mL)
and Water for Injection. The pH is 6.5.
1.2.Mechanism of Action
Rituximab kills cancerous B cells via three main mechanisms: complement-dependent cytoxicity
(CDC), antibody-dependent cell-mediated cytoxcity (ADCC), and apoptosis. CDC occurs when
a large group of plasma proteins (complement) work together to destroy invading pathogens and
malignant cells. Antibody-antigen complexes, such as the one between rituximab and CD20,
activate the complement. The complement protein C1 binds to the tail of the rituximab antibody
in a “lock and key” fashion and starts a series of reactions that creates a membrane attack
complex lining the B cell membrane and then creating a pore to allow the cellular contents to
escape and eventually die. ADCC is a process where the antibody-antigen complex forms and
then attracts other components of the immune system, including natural killer cells. The
receptors on these cells recognize and bind to the tail of the rituximab antibody. The natural
killer cells also carry granules filled with cytotoxic molecules. When the granules are released
after the natural killer cells bind with rituximab, they penetrate the cellular membrane of the B
cell and cause pores that facilitate the release of cellular content leading to the cell’s death. The
granules can also destroy the cells by attacking the nucleus. The final mechanism is known as

1. Introduction
apoptosis, or programmed cell death. Apoptosis is defined the death of cells that occurs as a
normal and controlled part of an organism’s growth or development. When rituximab bonds to
CD20 and forms the antibody receptor complex, it signals the cell to start the process. The
cytoskeleton collapses upon itself, the nucleus condenses, and the DNA fragments into small
pieces via enzymes. Membrane-bound vesicles are also shredded. At the end of apoptosis, the
cell has effectively destroyed itself. It is still unclear, however, whether these mechanism act
independently or in concert. Despite this, rituximab still proves to be an effective cure.
1.3.Properties
Sr.
No.
Property Value
1 Molecular Formula C6416H9874N1688O1987S44
2 CAS number 174722-31-7
3 Chemical Family Proteins
4 Melting Point Not applicable
5 Color Clear ,colorless liquid
6 Appearance
7 Molecular Mass 143859.7 g/mol
8 Boiling Point(degrees C) 100
9 Optimal pH range 6.5
10 Binding affinity for CD20
antigen
8.0 nM
11 Solubility in Water Soluble

1. Introduction
1.4. IMPACT
The impact that rituximab had is unprecedented. It provides a cure for curable lymphomas such
as diffused large cell lymphoma. The lifetime of patients with incurable lymphomas have been
extended. Recent numbers further demonstrate the success of rituximab. It has been considered
the top anticancer drug in the world since 2001. Sales in 2010 alone totaled $6.7 billion. Each
year, around 50,000 lymphoma patients are cured. Since its introduction in 1997 until 2010, over
two million patients have been treated. Prior to this discovery, there has been a long of stagnation
in finding cures or ways to extend lifetimes. Hopefully the success of Dr. Antonio Grillo-López
will inspire others to follow in his footsteps. He, himself, has said that he is neither a saint nor a
magician. Geduld led to his success, something that can be replicated by any ordinary person
with persistence and determination.
1.5. Manufacturers
1 Dr. Reddy’s Laboratories Ltd.(INDIA)
2 Genentech , South San Francisco.

2. Executive Summary
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 5
Product: RITUXIMAB
Installed Capacity: 100000 vials of 10 ml each with strength of 10 mg/ml.
Project Type: Greenfield.
Project Site: Raigad. State- Maharashtra
Raw Material Requirements:
Raw material Batch req ( kg) Rate (Rs/kg)
Annual
cost(Rs)
Inoc. Media Sol 129 368 47472
serum free media 246 18000 4428000
H3PO4 6275 8.5 53337.5
NaOH 5892 15 88380
Pro. A reg buff 3174 10 31740
Pro.A elution 5317 9.2 48916.4
Pro A equil 11562 9.2 106370.4
ION eq buff 1730 11.2 19376
ION wash buff 1732 22 38104
IOX el buff 97 18.5 1794.5
Nacl(1M) 1065 22 23430
ammonium Sulfate 74 480 35520
polysorbate80 2 110 220
sodium citratet dihydrate 0.25 2500 625
Total Raw material cost 4923286

Gross Cost of Production: 1437 Lakh of product produced.
Sales:
Selling Price (Average): 18000000 Rs/kg of Rituximab
Total Sales: 1800 Lakh/ annum.
Financing:
Total Fixed Capital Investment: 1302.2 lakhs
Debt: Equity Ratio: 1.5:1.
Term Loan: 808.45 lakhs @ 12 % per annum.
Promoter’s contribution: 274.87 lakhs (51% of equity)
Organizational participation: 80.84lakhs (15% of equity).
Public Issue: 183.25 lakhs (34% of equity).
Project Evaluation:
Break-Even Capacity: 40.28% (= 59.6 % of installed capacity.)
Payback Period: 3 year 7 months.
Return on Investment: 65.41 %
Weighted average cost of capital: 19.2%. (Desired return on equity = 30%)
Profitability Index: 1.18
Internal Rate of Return: 52.76 %

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.

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 .

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.

4.1. Medium preparation:
CD CHO medium is a protein-free, serum-free, chemically-defined medium optimized for the
growth of Chinese hamster ovary (CHO) cells and expression of recombinant proteins in
suspension culture. With a proven track record of quality for more than 10 years, CD CHO
medium contains no proteins or peptide components of animal, plant, or synthetic origin, as well
as no undefined lysates or hydro lysates. We import the CHO medium from Life Technologies.
4.1.1. DMEM and buffer preparation.
a) Materials
▪ DMEM (Dulbecco's Modified Eagle Medium): Gibco-Brl #12800-017, 1 pack for 1 L
Powder with high glucose with L-glutamine with pyridoxine hydrochloride with 110 mg/L
sodium pyruvate without sodium bicarbonate
▪ Sodium bicarbonate (NaHCO3): sigma # S 7277, 500 g (cell culture grade)
▪ 3X Distilled Water (D.W.)
▪ 0.22 µm vacuum filter (vaccucap 90): Gelman science #pn 4622-filterling capacity up to 10 L
CHRTGPH-3
Y-3
CHRTGPH-2CHRTGPH
Reaction-1
Batch reactor
Filtration
Reaction-2
Reaction-3

▪ DPBS (Dulbecco's PBS): sigma #D 5652, 10 bottles/pack, 1 bottle for 1 L
▪ Trypsin-EDTA (10X): sigma #T 4174, 100 ml
▪ FBS (Fetal Bovine Serum): JBI #S 001-01, 500 ml.
b) FBS
Serum supplies growth factors and nutrients. Serum requirement is dependent on cell type. Some
cell lines have been 'trained' to survive in medium with low serum. So, must check how much
serum amount your cells need before starting cell culture.
These are the serum variables you must consider.
- The percentage of serum: Most cells require 5-20% in the medium for good growth (all of our
cell line require 10% FBS).
- The types of serum: Some cells like horse serum or calf serum, but our system use fetal Bovine
serum (FBS/FCS).
- Heat-inactivate serum: Serum in subjected to heat to inactivate components such as
complement.
To heat-inactivate serum
1) Thaw the frozen serum (company supply FBS in -20℃) at 37℃ for 5-6 hours.
2) Incubate the thawed serum at 56-65℃ for 30 min (shake the bottle gently in every 10
min)
3) Aliquot the serum in 50 ml conical tube and then seal with parafilm.
4) Freeze and store. Thaw aliquots in a 37℃ water bath as needed.
(Caution !!)
Serum is very expensive. Always aliquot and freeze serum, and add it to medium just before
use. Store unused portions of thawed aliquots in the refrigerator, where it will be fine for

several weeks…Please do not waste serum!!!
c) Preparation of DMEM
* History of culture media
Complete media range in complexity from the relatively simple Eagle's MEM [Eagle, 1950] -
Complex to Medium 199 [Morgan, 1950], CMRL 1066 [Paker, 1957], MB752/1 [Waymouth,
1959], RPMI 1640 [Moore, 1967], and Ham-F12 [Ham, 1965].
The complex media contain a large number of different amino acids, additional vitamins and
Extra metabolites in F12 (optimizing by cloning) and in Dulbecco's modified Eagle's MEM
(DMEM) [Dulbecco, 1959], DMEM-F12 [Barnes and Sato, 1980].
1) Measure out 5% less 3X D.W. than desired total volume of media.
2) Add powdered media to D.W. at RT with gentle stirring (Do not over-stir or heat).
3) Rinse remained powder in the package with D.W. and add it to media.
4) Add 3.7 g of NaHCO3 to make 45mM concentration/L of media.
5) Add D.W. to desired volume. Stir until dissolved (Do not over mix).
6) The final pH of the media should be pH 7.4. Adjust pH of media to 0.2-0.3 below the
Desired working pH (~ 6.95) using 1 N NaOH or 1 N HCl (generally HCl). Add slowly
With stirring. The pH units will increase 0.1-0.3 upon filtration. After pH has been adjusted,
Keep container closed with aluminum foil until media is filtered.
7) Sterilize immediately by using a Gelman vaccucap 90 through dispense in 500 ml bottles.
8) Store media at 4℃ in the dark until use. .

4.2. Media for Cell Culture
We are now using washable and reusable glass bottles to prepare our media and our PBS
solutions. The media bottles have tops which are compatible with the disposable sterile filters
from VWR. These bottles should be washed with bleach and water (3x with bleach and 9x with
water), then dried and autoclaved. After being autoclaved you can spray the outside with EtOH
and bring into the laminar flow cabinet. I would recommend opening the bottle and placing the
lid upwards on the bottom of the cabinet and turn on the UV light for 10 minutes just to be sure.
4.2.1 CHO K1 (-) Media
This media is suitable for non-transfected CHO K1, or transiently transfected CHO cells which
do not have any antibiotic resistance.
To prepare the media will need the following:
1. 430 mL of DMEM
2. 50 mL of heat inactivated FBS
3. 5 mL of Pen/Strep
4. 5 mL of Non-essential amino acids
5. 10 mL of L-glutamine
Mix all reagents together and run through a sterile filter.
4.2.2 CHO K1 (+) Media
This media is suitable for CHO K1 cells which stably express fluorescent proteins and are
resistant to geneticin.
To prepare the media you will need the following:
1.400 mL of DMEM – Low Glucose
2.50 mL of FBS

3.5 mL of Pen/Strep
4.5 mL non-essential amino acids
5.10 mL of L-glutamine
6.0.235g of geneticin
The geneticin is not immediately soluble in the DMEM. It is recommended that you add the
geneticin first to the warm DMEM to increase the solubility and maximize the concentration of
geneticin in the sample.
4.3. Bioreactor media composition
The composition of the fermentation media is as follows:
Table 4.1: Media Composition
SR.NO. COMPONENT
1 Amino Acid(20)
2 Vitamin (9)
3 Organic Compound(8)
4 Inorganic Salts
The pH of the media is maintained at 6.5 using NaOH dosing and the temperature is maintained
at 37 0
C.
4.4 Chromatography:
This is very common separation process that is used in many different industries. Small
resin beads, typically agarose or polyacrylamide, contain surface properties that allow for the
binding of specific molecules. These can either be molecules of interest, or the impurities that
need to be removed. In this case of the former, a solution containing the protein of interest is
pumped through resin, resulting in the protein binding to the resin. There are very specific
condition, such as the pH and polarity of the solution, that allow this interaction to take place.

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:

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.

5. Kinetics and thermodynamics of the process
5. Kinetics and thermodynamics of process
5.1. Kinetics
The general aim in a biological reaction is to support the growth of a specific organism and to
encourage a high product yield. It is therefore common practice to limit the concentration of
essential nutrient to give controlled overall growth while provide others in excess. In this case,
glucose is taken as the limiting reactant.
All kinetic data were submitted to curve fitting techniques. An appropriate polynomial function
was fitted by the least squares method to the measured concentration data. The derivative with
respect to time was then calculated for the values obtained with the help of the polynomial
function. The specific consumption or production rates could then be calculated by dividing the
derivative by the viable cell concentration (Xv) at selected time points.
The apparent specific growth rate, µ, was calculated using the following equation:

The production of Rituximab belongs to category of fermentation wherein the production is due
to recombinant technology and cell culture production of Chinese Hamster Ovary primary but
the reaction rate is complex.. Hence proper conditions and control is required for maximum
yield. Below the growth trend and substrate consumption for the CHO is given.
Kinetics of CHO cell growth,
Fig.1. Kinetics of CHO cell growth, in 96 well plates containing 200 mL of different
media: serum-free reference medium (*), serum-free medium supplemented with
25 mg/L of sinapic acid (&), serum-free medium supplemented with 4 g/L of
rapeseed peptide fraction (~) and serum-free medium supplemented with 25 mg/L
of sinapic acid and of 4 g/L of rapeseed peptide fraction (_). Experiments were
performed in triplicate

5.2. Thermodynamics
Biochemical pathways are complicated and it is not possible to find the ΔG values for every
reaction taking place. But we can safely conclude that the overall reaction is thermodynamically
feasible.

6. Process Block Diagram
Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 20
InoculumMedium
Preparation
Air
sterilization
Bioreactor Centrifugation Protein A
Chromatography
Ion Exchange
Chromatography
Cell mass
Nutrient
Air
VIALS FILLING &
PACKING
Diafiltration
FormulationStorage
VR filtration
Final Filtration

7. Site Selection
7. Site Selection
Proper site selection is one of the factors that govern the success of any project. The entire site
selection exercise can be divided into three main factors:
 Profitability factors: profitability of project
 Prosperity factors: prosperity of nation
 Productivity factor: productivity of plant
7.1. Profitability factors
7.1 .1.Availability of raw materials:
Raw material contributes a major share to the operating cost of the project. This in turn is going
to determine the cost of production of the product eventually deciding the profit margin. So it is
important to consider this component in site selection.
7. 1.2. Market for the Product:
The product Rituximab is used for cancer treatment, Hence the manufacturing site should be
close to metro cities so that transportation cost is reduced
7.1.3. Land:
Adequate land space must be available for all the buildings, units and equipments. Also
provision for any future growth has to be considered. Other space requirements like effluent
treatment plant or green belt also should be given due consideration. This land must be available
at affordable price as this would eventually be contributing to the fixed cost of the project.
7.1.4. Soil Assay:
This includes the type of soil, its bearing capacity, and identification of seismic zone and height
of water table. Proper soil survey to avoid the mechanical breakdown of the plant is necessary.
7.1.5. Climatic conditions:
The necessary climatic data like average rainfall, minimum and maximum temperature,
frequency of cyclones and hurricanes should be considered while selecting a site. These have
effect on the fixed cost of the plant.

7. Site Selection
7.1.6 .Power:
Regular and uninterrupted power supply at conceded rates is an essential factor to run the project
smoothly.
7.1.7 .Water quality and availability:
This is the most important parameter for site selection since water is required in huge quantities
for the process. Water must be available in adequate amounts and with good quality at cheaper
rates. Choosing a site which guarantees the uninterrupted supply of water having compatible
qualities at affordable rate is desired.
7.1.8. Environmental considerations:
Having a common effluent treatment plant in an industrial complex always helps reduce the load
on the project.
7.2. Productivity factors
7.2.1 Communication:
Communication facilities like Telephone, Fax, Telex, e-mails, etc. should be there at the site
location.
7.2.2 Labour:
Availability of skilled labour is important.
7.2.3 Infrastructure:
The existence of well developed infrastructure is desirable. The site should easily accessible by
road and railways. Also general civic amenities should be easily accessible.

7. Site Selection
7.3 Prosperity factors
7.3.1 Appropriate Utilization of Raw materials:
The natural resources of the country have to be utilized appropriately and efficiently. There
should be proper utilization of resources, decreasing the load on the public transportation system.
7.3.2 Dispersal of Industries:
Utilization of most of the available land, coupled with the objective of de-industrialization of
metropolitan cities is an objective to be fulfilled by dispersal of industries away from residential
zones which has lead to the development of this industrial estate. This calls for choosing a site
which is located in specially secured industrial zones so as to take advantages of the facilities
already present and keep distance from populated areas.
7.3.3 Security of the Nation:
The site should not be situated in a politically sensitive area, so it does not endanger national
security.
7.4.Site evaluation
Based on the above mentioned factors the two sites which seem suitable to set up the
manufacturing unit are Hosur, Krishnagiri district under TIDCO(Tamil Nadu Industrial
Development corporation) (Site A) and Raigad under MIDC(Maharashtra Industrial
Development corporation )(Site B).Both of these sites have been declared as biotech SEZ’s by
the respective governments.
Table 1. 1. Site Evaluation
Criteria Site A Site B
Land and site development 8 7
Building and civil
construction
7 7
Climate 8 8
Raw Material source 8 10
Product Market 6 9

7. Site Selection
Water/Effluent treatment 8 6
Power 6 8
Environmental Consideration 8 6
Housing and social
community factor
7 8
Staff transport 5 8
Equipment transport 8 9
Taxes and subsidies 7 8
TOTAL 86 92
Therefore Raigad, Maharashtra is chosen as the manufacturing site.

8. Material Balance
8. Material balance
A] Inoculum preparation
The inoculum is initially prepared in 225 ml T-flasks. The material is first moved to roller bottles
(25 L), then to 50 L and subsequently to 75 L disposable bag bioreactors. Sterilized media is fed
at the appropriate amount in all of these four initial steps (3.6, 11.4, 43.6, 175.4 kg/batch
respectively). The broth is then moved to the first (25 L) and second (50 L) seed bioreactor. For
the seed bioreactors the media powder is diluted using WFI in one prep tank.
B] Bioreaction section
Serum-free low-protein media powder is dissolved in WFI in a stainless steel tank (MP-103).
The solution is sterilized using a 0.2 µm dead-end polishing filter (DE-103). A stirred-tank
bioreactor (PBR1) is used to grow the cells, which produce the therapeutic monoclonal antibody
(Rituximab). The production bioreactor operates under a fed batch mode. High media
concentrations are inhibitory to the cells so half of the media is added at the start of the process
and the rest is fed at a constant rate during fermentation. The concentration of media powder in
the initial feed solution is 11.73 g/L. The fermentation time is 12 days. The volume of broth
generated per bioreactor batch is approximately 50 L, which contains roughly 0.5 kg of product.
Basis: Per batch
Total working days = 300 days
Total Product to be produced= 10 Kg (10mg/ml 10ml fill 100,000 vials)
So 10 Kg to be produced in 300 days
Total batches = 20
1 batch production = 0.5 kg Rituximab

8. Material Balance
8.1. Bioreactor B001
Volume = 25 Liters
Product Con = 11.73 g/l/day
Total mass of cell = 11.73×V = 20×25=293.75g
Total mass of cell in 1 batch ( 6 days) =1762.5g = 1.76 g
The size of B001 is 34 Lit with 25 L working volume.
Serum free media supplement 4.2 g/lit
Total serum free media supplement = 4.2 × 25=105 g.
Table 8. 1:Across B001
input(kg)(1) input(kg)(2) output(kg)(3)
Cell mass 0.5 1
Serum free media 0 0.105 0
Volume = 50 Liters
Product Conc. = 11.73 g/l/day
Total mass of cell = 11.73×V = 11.73×50=586.5g.
Total mass of cell in 1 batch ( 6 days) =6000 mg = 6 g
B001
1
3
2
2

8. Material Balance
The size of B002 is 65 Lit with 50 L working volume.
Serum free media supplement 4.2 g/lit
Total serum free media supplemet =4.2 × 50=210 g.
Table 8. 2: Across B002
input(kg)(3) input(kg)(4) output(kg)(5)
Cell mass 1× 10-3
0 1.5 × 10-3
Serum free media 0 210 0
8.3. Material balance across the Production Bioreactor B003
The size of the production Bioreactor B001 is 0.065 m3
with 0.05 m3
working volume. Serum-
free low-protein media powder is dissolved in WFI in a stainless steel tank. The solution is
sterilized using a 0.2 µm dead-end polishing filter. A stirred-tank bioreactor is used to grow the
cells, which produce the therapeutic monoclonal antibody (Rituximab). The production
bioreactor operates under a fed batch mode. High media concentrations are inhibitory to the cells
so half of the media is added at the start of the process and the rest is fed at a constant rate during
fermentation. The concentration of media powder in the initial feed solution is 42 g/L. The
fermentation time is 12 days. The volume of broth generated per bioreactor batch is
approximately 80 L, which contains roughly 50 kg of product.
B002
3
5
4
2

8. Material Balance
Total cell mass=75 ×11.73=879.75g=879.75 g
Amount of serum free media=4.2×75=315g. =0.315Kg.
Amount of media required for composition = 0.8039
Time taken for it to get completely consumed
Thus after every 7.14hrs, 0.8039kg of media needs to be supplied.
SR.NO. COMPONENT mg/L(x) Conc (75*x) Kg
1 Amino Acid(20) 476.4 0.035
2 Vitamin (9) 3.6 270 × 10-6
3 Organic Compound(8) 1948.61 0.146
4 Inorganic Salts 8300.9 0.622
Total - 0.8039
Table 8.3: Across B003
input(kg)(5) Input(kg)(6) output(lit)(7)
Amino Acid(20) 0.035 0 -
Vitamin (9) 270 × 10-6
0 0
Organic Compound(8) 0.146 0 0
Inorganic Salts 0.622 0 0
Cell mass 0 879.75× 10-3
Serum free media 0 0.315
Total volume(lit) 0 0 72
B003
5
7
6
2

8. Material Balance
8.3. Material balance across the Centrifuge C001
Between the downstream unit procedures there are 0.2 μm dead-end filters to ensure sterility.
The generated biomass and other suspended compounds are removed using a Disc-Stack
centrifuge. During this step, roughly 2% of Mab is lost in the solids waste stream resulting in a
product yield of 98% for 3.8 hrs.
Table 8.4: Across C001
input(7) output(8) output(9) % yield
Mass of cell (g)
841.97 15.24 826.73 98
Volume(Lit) 71.78 1.3 70.48 98
8.4. Material balance across the Affinity Chromatography Column F001
The bulk of the contaminant proteins are removed using a Protein-A affinity chromatography
column (C-101). The yield on Mab for this step is 90%.The protein solution is then concentrated
5x and diafiltered 2x (in P-21 / DF-101). The yield on product is 98% and this is represented by
the product denaturation feature of the Diafiltration operation. The concentrated protein solution is
then chemically treated for 1.5 h with Polysorbate 80 to inactivate viruses (in P-22 / V-111).
C0017 9
8

8. Material Balance
Table8.5: Across F001
Mass of cell (g) 826.73 41.28 800.22 95
Volume(Lit) 70.48 3.52 68.22 95
8.5. Material balance across the Ion Exchange Chromatography Column F002
An Ion Exchange chromatography step follows (P-24 C-102) with a yield on Mab of 90%.
Ammonium sulfate is then added to the IEX eluate (in P-25 V-109) to increase the ionic
strength for the Hydrophobic Interaction Chromatography (P-26 C-103) that follows. 20% of
Mab is lost during the HIC procedure.
F001
9 11
10
F00211 13
12

8. Material Balance
Mass of cell (g) 785.44 78.47 706.96 90
Volume(Lit) 66.96 6.69 60.27 90
8.6. Material balance across Virus Retentive Filtration F003S
Mass of cell (g ) 706.96 70.61 636.35 90
Volume(Lit) 60.27 6.02 54.25 90
8.6. Material balance across Difiltration F004
F00313 15
14
F00415 17
16

8. Material Balance
Mass of cell (g ) 636.35 31.78 604.56 95
Volume(Lit) 54.25 2.71 51.54 95
8.7. Material balance across Formulation Column FC001
Table 8. 9: Across FC001
input(gm) output(Kg)(20)
Polysorbate-80(17) 37.97 0
Sodium Citrate dehydrate(18) 399.47 0
Sodium Chloride(19) 488.25 0
Cell mass(20)(g) 604.56 0.592
FC001
17
3
P00210 13
12
21
17
19
P00210 13
12
18
P00210 13
12
20
P00210 13
12

8. Material Balance
8.8. Vials Filling
Table8.10: Across V001
input Output Output % yield
Mass of cell (g ) 592.36 5.86 586.5 99
Volume(Lit) 50.50 0.50 50 99
8.8. Summary of the entire process
The material balance summary across all the units in the process is as follows
Table8.11 : Material balance summary
Purification Step %recovery at every step
Centrifugation 98
Affinity Chromatography 95
Ion Exchange Chromatography 90
Virus retentive filtration 90
Dilfiltration 95
Formulation 99
Packing and storage 99
Total Process efficiency 69.64

9. Energy balance
9. Energy balance
9.1. Across production Bioreactor B003
For sterilizing the Bioreactor and the Bioreactor media, it has to be heated to 121°C, be held at
that temperature for a certain length of time and cooled to 37°C, the temperature at which
fermentation takes place. Thereafter, fermentation being an exothermic process the temperature
has to be maintained at 37°C by passage of cooling water through the coils
9.1.1. The heating cycle
The vessel is heated from room temperature to 121°C by using saturated steam at 4bar pressure.
The properties of steam at 4 bar are
: temperature difference:121-30=91°C=365 k
Cp: Specific heat capacity of Bioreactor media: 4.18 kJ/kg/°K
m: mass of the substrate media:72 kg
:Latent heat of condensation of steam at 4 bar: 2132.95 kJ/kg
9.1.2.The cooling cycle
Cooling water at 28°C from the cooling tower is passed through the coils placed inside the vessel
to cool it from 121°C to the Bioreactor temperature of 37°C.
:Specific heat capacity of cooling water:4.18 kJ/kg
: difference in cooling water inlet and outlet temperature:5°C
:temperature difference of the Bioreactor media:121-37=84°C

9. Energy balance
9.1.3.Maintaining of Bioreactor temperature at 37°C
Bioreactor is an exothermic process. The metabolic heat generation rate is assumed to be 10
kW/m3
(Biotranformation and bioprocess,pg 182).The heat removal rate should be 115 kW.
Therefore, flow rate of cooling water required is 5.5 kg/s
9.5. Across fired heater E001
Air at room temperature of 30°C and 40 % relative humidity is heated by passing through the
tubes of coal fired heater to 110°C
CV: Calorific value of coal :20,000kJ/kg.

10. Equipment Design
10. Equipment design
10.1. Process Design of Bioreactor
10.1.1. Mode of operation
As can be seen in Section of material balance intermittent supplies of media is required to
maintain the cell growth composition. Since it is aerobic fermentation, continuous supply of
oxygen is essential. Considering the difficulty of controlling and maintaining monosepsis
condition in a continuous mode, fed batch mode is used.
10.1.2. Bioreactor type
The type of Bioreactor depends on the nature of the process (including cell kinetics), operating
conditions (namely, mode of operation and gas liquid flow patterns), and physical and chemical
properties of the substrates and the microbe. For the required production capacity, volume of
substrate media necessary in the bioreactor is 0.075 m3
.For this scale of operation a mechanically
stirred tank reactor consumes less power per unit volume as compared to non-mechanically
agitated system.
10.1.3. Bioreactor geometry (Walas,1990, Pg no .288)
Assuming liquid medium occupies 75% volume
Volume of the Bioreactor:
The height to diameter ratio for the vessel is taken as 2 because
 The system has low viscosity(<25 Pa s)
 The volume required for fermentation is not very huge
 The system is shear sensitive. Hence less number of impellers should be used



10.1.4. Impellor type and design
The functions to be performed by the impeller in the system are
 Mixing
 Effective dispersion of air
The controlling parameter is the oxygen dispersion in there for which radial impellers are
preferable. However they consume high power. Hence we use a mixed flow pitched blade
turbine impeller. Let the d:D be 1:13 and W:D be 1:5
For the CHO cell under the process conditions of 72 L substrate volume (Dtank=215mm) and 500
rpm impeller (3 six blade disc turbine dimp=80mm) speed, the air flow rate for optimal production
is found to be 1 vvm (El Enshasy ,H. et al,2008)
( )
During scale up ,the objective is to maintain the same mass transfer coefficient of oxygen.
For pilot scale
At this calculated , is 1.37
( )
6.515W
s

For production scale
( )
s
10.1.5.Sparger design
Gas hold up in the system (Handbook of chemical engineering calculations, Pg no. 575)
( )
= volumetric flow rate of air = 0.2 m3
/s
Notations:
D: Diameter of tank
d: Diamter of impeller
W: Width of impeller
: density of the medium
: Volumetric air flow rate
N : speed of impeller
: gas hold up in the system
V :volume of the Bioreactor.

10.2.Protein A CHROM COLUMN
Protein A is a cell wall component produced by several strains of Staphylococcus aureus. The
46.7kDa-protein consists of a single polypeptide chain that is essentially devoid of carbohydrate.
Native Protein A has contains four high-affinity (Ka = 108
/mol) binding sites that are capable of
interacting with the Fc region of IgG-class antibodies from selected mammalian species. IgG-
binding function is optimal at pH 8.2, but efficient binding also occurs in neutral and
physiological buffers (pH 7.0 to 7.6).
 Native Protein A – immobilized Protein A is ideal for polyclonal IgG purification.
 Agarose resin – support is crosslinked 6% beaded agarose (CL-6B), the most popular
resin for protein affinity purification methods.
Properties of crosslinked 6% beaded agarose (CL-6B):
 Support pH Stability: 2 to 14 (short term); 3 to 13 (long term)
 Average Particle Size: 45 to 165 microns
 Exclusion Limit: 10,000 to 4,000,000 daltons
 Maximum Volumetric Flow Rate: approx. 1mL/minute.
 Maximum Linear Velocity: 30cm per hour
 Maximum Pressure: less than 1.5 bar, defined as the maximum pressure drop across a
column that the resin can withstand (Note: The indicated gauge pressure of a liquid
chromatography apparatus may be measuring the total system pressure rather than the
pressure drop across the column.)
 Inert and stable – superior manufacturing method immobilizes Protein A by charge-free,
leach-resistant covalent bonds, resulting in low nonspecific binding and enabling multiple
uses without decline in yield
 High capacity – this “Plus” variety of Pierce Protein A Agarose has a dense load of
immobilized Protein A, providing a binding capacity greater than 34mg human IgG/mL
resin (approx. 16 to 17mg mouse IgG/mL resin)

 Multiple formats – choose from bottled resin slurries, centrifuge-ready columns,
complete purification kits, two sizes of FPLC-ready chromatography cartridges, and 96-
well filter plates.
10.3. Process Design of Diafiltration Unit
Is a technique that uses basic principles of filtration to completely remove, replace or lower the
concentration of salts or solvents from solutions containing biomolecules, Uses permeable
membrane to separate the components mainly based on size and column-based gel filtration.

10.3.1. Design Consideration
a) Type of flow (tangential vs. direct)
Direct Flow:
- Large molecule trapped on membrane and forms gel
- More susceptible to fouling
- Flux rate decreases as volume filtered increases

Tangential Flow
- Solute diffuses through the surface of the membrane tangent to the flow of the feed
- Minimize buildup of molecules – less fouling
- Prevents rapid decline in flux rate.
Reference:-Millipore Inc., 2003
b) Membrane selection
Primarily based on size of biomolecule and Molecular weight cut off (MWCO) of the
membrane should be 1/3rd
to 1/5th
of the MW of the molecule to be retained,Typical
MW of mAb: 150kDa => 30000 MWCOO,For protein separation: 30 LMH.
c) Type of diafilter modules
i) Flat sheet tangential flow
ii) Hollow fibre
iii) Tubular
iv) Spiral wound

d) Continuous vs. discontinuous flow
Continuous
 Typically constant volume
 Removal rate of salt = addition rate of water
 Addition of WFI is at 1/3rd
of the removal rate of salt (filtrate).
 More suited for process scale- requires pumps
Discontinuous
 Concentration and dilution cycles
 Usually more feasible on a laboratory scale

9.2.2. Final design
Feed flow rate = 3L/min/m2
-Hollow fiber cartridges
Membrane area needed
Mem. area = filtrate vol / (filtrate flux * process time)
= 50/(30x2)=0.83 m2
Pump Feed rate (L/min) = Feed flow rate X Area
= 3 X 0.83=2.49 L/min
-UNIT NUMBER:
Stainless stain housing
88
90
92
94
96
98
100
102
15
17
19
21
23
25
27
29
0 1 2 3 4 5 6
PercentofAceticAcidremoval
MembraneArea(m2)
Diafiltration Volume
Effect of diafiltration volume on membrane area
requirement and percent of acetic acid removal
Membrane Size Removal of Acetic Acid

Steam in place cartridges can be added
Reference :- GE Healthcare (2007)

11. Mechanical design Of Bioreactor
11. Mechanical Design of Bioreactor B003:
H =0.41m D= 0.39m
11.1. Thickness of vessel required
Max pressure P = 1.013 bar during cell growth..
Hence design pressure =1.013 *1.4= 1.418 bar = 0.141 N /mm2
t
= 0.141× 390 / ( 2× 140 × 0.85)-0.141) + 2
= 2.23 mm
We take thickness 6mm as this must be same as head thickness.
11.2. Design of Heads:
At the top:
A torispherical head is used . It is connected to the shell by means of a flanged joint.
Crown Radius Rc = 390 mm
Knuckle Radius R1 = 24 mm (6% of Rc)
The thickness of the torispherical head is given as
2.f.J
.WP.R
t
c
h  + C
where W = stress intensification factor
]
R
R
3[
4
1
1
c

= 1.77

J = joint efficiency = 0.85 ( flange joint)
 th = 0.41+2 = 2.41 mm
At the bottom:
A torispherical head is welded to the shell at the bottom end.
Here J = 1
Applying the same formula , thickness = 3.12 mm ,
So we use 4 mm thickness
11.3. Design of Flanges for Head and Shell
Internal gasket diameter, Gi = 390 +10 = 400mm
External gasket diameter is calculated as;
pmpY
mpY
G
G
a
a
i
o



Gasket seating stress, Ya =52.5 N/mm2
Gasket factor (m) =3.75
0013.1
i
o
G
G
Go = 400.54mm
A flat asbestos gasket of 390 mm internal diameter and 400 mm external diameter is used.
Gasket seating width = b = (400-390) ×0.5 = 5
Under atmospheric conditions bolt load due to gasket reaction is given by:

Wm1 =  b G Ya
Where G = ( Gi + Go)/2 = 800.54 mm
Gasket seating stress, Ya =52.5 N/mm2
 Wm1 = 6.6 x 105
N
The load under operating conditions is given by
Wm2 = .P.G
4
b).G.m.P2.( 2
 
= 8.42x 104
N
The total bolt area is calculated on the basis of the greater load
 A =
f
Wm1
where f is the permissible stress in bolt (138 N/mm2
)
Therefore, A = 41,826 mm2
Let us use bolts of area =500 mm2
Hence number of bolts required =78.
Bolt area = 78*d
4
2
Hence bolt diameter = 28.56 mm
So we use M26 bolts ( 78 Nos )
Pitch circle diameter (P.C.D) = Outside diameter of gasket + 2  diameter of bolt + 12mm
= 400+ 2*28.56 +12 = 469.12 mm

Flange thickness is given by
fK
P
Gtf 
where
HG
hW5.1
3.0
1
K
gm


Wm = total bolt load = 6.6 x 105
N
hg = Radial distance from gasket load reaction to basket circle
= (PCD - G)/2
= 34.5 mm
H = Total hydrostatic end force
= PG
4
2
= 0.6 x 106
N
K = 2.69
tf = 44.6 mm
Use 50 mm thickness of flange.
11.4. Design of Nozzles:
The nozzles provided are as follows:
1) On shallow dished head at top:
 200 mm nozzle for substrate inlet.
 50 mm nozzle for pressure indicator
 50 mm nozzle for pH indicator

 50 mm nozzle for thermocouple
 50 mm nozzle for acid inlet
 50 mm nozzle for antifoam
 50 mm nozzle for Pressure relief valve
 5 mm nozzle for level detector
 450 mm nozzle for manhole.
 150 mm nozzle for sight glass.
2) On torispherical head, at the bottom:
 20 mm nozzle for draining out broth
 10 mm nozzle for Air inlet
3) On shell:
 30 mm nozzle for coil inlet
 30 mm nozzle for coil outlet
For 30 mm nozzle on shell:
Nozzle thickness required,
tn =
P-2fJ
dP i
=0.154*300/(2*1*140 - 0.154)
= 0.165 mm
Actual thickness taken =6 mm.The area for which compensation is required is
A = d ts`
= 300 x 1.42
= 426 mm2

Area available for compensation:
i) Area of compensation provided by the portion of the shell as excess thickness
Ah = d (ts – ts`)
Where
ts` = theoretical shell thickness required = 1.42 mm
ts= Actual shell thickness used = 6mm
d = Diameter of nozzle
As = 1374 mm2
ii) Area of compensation provided by the nozzle:
An = 2 x Ho x (tn - tn`)
tn = Actual thickness of nozzle used = 6 mm
tn` = Nozzle thickness required theoretically =0.165 mm
Ho = 2.5 x tn
An = 29.175 mm2
As + An =1181.175 mm2
> A
So reinforcement ring is need not be provided.From similar calculations, it is found that no
reinforcement ring is required for any of the nozzles.
11.5. Design of supports
Selection of support:
Vertical vessels : Bracket or Skirt support
H/D: 2 to 5 - Bracket support

H/D>5 – Skirt support
Hence, we use Bracket support
No. of brackets for D< 3000mm = 4.
i) Thickness of stiffener ( horizontal plate)
Wmax = Σ W / no. of brackets (neglecting wind loads as vessel is indoors and is not
very tall)
= (11.5*1200)*9.8 /4
=33,810 N













 44
4
max
*
7.0
Lb
b
t
L
lb
W
f
h
h
Where,
fh = permissible bending stress = 155 N/mm2
l= 75 mm
b=h=200 mm
L= 300 mm
Hence , th = 0.51mm
th = 2 mm
ii) Thickness of Gusset plate


COShf
lW
t
h
g 2
max3

Θ = 45 o
Hence tg = 1.22 mm
We take tg = 2 mm
11.6. Design of shaft :
Diameter of agitator = 700 mm
Agitator Speed = 151 rpm
Power = 3.6 kWatt
The continuous average torque on the shaft is given by,
Tc= power / (2xxN),
= 229.18 N-m
The shaft must be capable of resisting 1 ½ times the continuous average torque
Tm = 1.5 x Tc
= 344 N-m
= 34400 N –cm
Zp =
s
m
f
T
fs = Maximum permissible shear stress on shaft ( 9457 N / cm2
)
Zp = Polar modulus of section of the shaft
Zp =
16
3
d
(×d3
)/16= 3.63cm3
d = diameter of shaft

 d = 2.65cm
Hence, we use a shaft of 6 cm diameter.

12. Equipment Sizing
12. Equipment sizing
To ensure 5% by volume inoculum at every stage of fermentation, the size of B001 is taken as
0.025 m3
.
D:Diameter of tank h:Height of tank
Table 11.1 :Sizing of B001
Type Jacketed stirred tank vessel
Operating temperature 370
C
Operating pressure 1 atm
Volume 0.025m3
Diameter 0.3m
Height 0.3m
MOC SS316
12.2.Seed Bioreactor B002
The sizing is done as described in Section 11.1
Table 12.2:Sizing of B002
C
Volume 0.05m3
Diameter 0.4 m
Height 0.4 m
MOC SS316

12.3.Production Bioreactor B003
The sizing is done as described in section 11.1
Table 12.3 : Sizing of B003
C
Operating pressure 1atm
Volume 0.075 m3
Diameter 0.5m
Height 0.5m
MOC SS316
12.4.Centrifuge C001
The quantity of supernatant to be processed is 0.07 m3
.
Let the flowrate through the centrifuge be 0.25m3
/hr(Perry’s).Time taken for complete
centrifugation= . Settling velocity in presence of centrifugal force
according to stokes law
:settling velocity of particle under gravity (m/s): m/s
G: ratio of settling velocity under centrifugal force to that under gravity: 4300(assumption).
:diameter of cell particle(m):2µm
:density difference between cell particle and water:600kg/m3
:Vicosity of supernatant(Pas):8.5× Pa s
Area required for centrifugation=

Table 12. 4. Sizing of C001
Type Disc stack centrifuge
Operating temperature 30º
C
G 4300
Area 53.41 m2
Diameter 0.5m
No.of discs 12
MOC SS316
12.5. Protein A CHROM COLUMN F001
Type Protein A CHROM COLUMN
Column Agarose resin, 4.6mm ID × 10cm
Eluent A] 20mmol/L MES buffer, pH 6.0
B]0.5mol/L NaCl in 20mmol/L MES buffer, pH 6.0
Gradient 0min (10%B), 15min (30%B), linear
Flow rate 1.0mL/min
Temperature 250
C
Injection volume 20μL
Samples A: human antibody, IgG1
B: human antibody, IgG1
Sample concentration 0.5g/L

12.6. ION EXCHANGE COLUMN F002
12.6.1 Anion Exchange column STREAMLINE Q XL, 7.5 l
Certificate of Analysis Yes
Flow Velocity <500 cm/h
Matrix 6% cross-linked agarose containing a quartz core with dextran surface
extender
Storage Conditions 4 to 30°C, 20% Ethanol
Chemical Stability All commonly used buffers, nonionic detergents, 1 M NaOH, 6 M guanidine
hydrochloride
Ligand Quaternary amine
Ion Exchanger Type Strong anion exchanger
Ionic Capacity 0.23-0.33 mmol Cl-
/ml medium
pH Stability Cleaning-
in-Place (CIP)
2-14
BioProcess Medium Yes
Average Particle Size 200 µm
Binding Capacity/ml
Chromatography
Medium
> 110 mg BSA/m medium
pH Stability Working
Range
2-12
Particle Size 100 µm-300 µm

12.6.2. Cation Exchange column
STREAMLINE SP XL
Chemical Stability All commonly used buffers, nonionic detergents, 1 M NaOH, 6 M guanidine
hydrochloride
Flow Velocity <500 cm/h
Storage Conditions 4 to 30°C, 20% Ethanol + 0.2 M Sodium Acetate
Ionic Capacity 0.18-0.24 mmol H+
/ml medium
Average Particle Size 200 µm
Matrix 6% cross-linked agarose containing a quartz core with dextran surface
extender
Ion Exchanger Type Strong cation exchanger
Particle Size 100 µm-300 µm
Binding Capacity/ml
Chromatography
Medium
> 140 mg lysozyme/ml medium
Ligand Sulphopropyl
pH Stability Cleaning-
in-Place (CIP)
3-14
pH Stability Working
Range
4-13
Certificate of Analysis Yes
BioProcess Medium Yes

12.7.Virus retentive filtration F003
The product concentration in the system =11.73 g/L.It is concentrated 5 times.An optimum flux
of 0.000025m/s is obtained at a trans membrane pressure of 72.4kPa.Let process take 4 hrs.
Table 12.5 .Sizing of Filtration F003
Type Tubular membrane unit
C
Transmembrane pressure 72.4 kPa
Area 1.64m2
No of modules 16
MOC Regenerated cellulose membrane , SS304
cover
12.8. Diafiltration Unit F004
The protein concentration in the system 2.5 wt %(24.62 kg/m3
).It is concentrated 5 times. An
optimum flux of 0.00025 m/s is obtained at a trans membrane pressure of 72.4kPa.
Table12.6.Sizing of Filtration P004
Type Tubular membrane unit
C
Trans membrane pressure 72.4 kPa
Area 6.56m2
No of modules 58
MOC Polysulfone membrane , SS304 cover

12.9. Air Compressor S0001
Compressed air is required for fermentation.
Air flowrate:70.92m3
/hr
Air inlet pressure:1.5 bar
Table 12.7. Sizing of S0001
Type Single stage axial flow compressor
Power 4HP
Capacity 0.02 m3
/s
Suction pressure 1 bar
Discharge pressure 2bar
MOC SS304
12.10.Pumps.
Different pumps are used at various stages in the process. Each pump is attached with a storage
vessel for liquid. They are as follows
Table 12.8. Sizing of pumps
Function Capacity(m3
/hr) Discharge
pressure(bar)
Power(HP) Type
P001 2.6 1 0.096 Centrifugal
P004 0.138 1.724 0.00885 Centrifugal
P005 0.025 1.724 0.0016 Centrifugal
P007 0.00483 1.724 0. Centrifugal

13. Instrumentation and Process control
13. Instrumentation and process control
In any process, instrumentation and control plays an important role to help achieve the desired
degree of productivity. In a bio-fermentation process, it is important to maintain certain
parameters at a specific value for the optimum growth of cells. The basis control strategy
involves a sensor which measures the process variable and converts it into appropriate signal via
the transmitter .The transmitter then sends the signal to an indicator and controller to which set
point is fed for the controlled variable. The indicator and controller compare the values and
depending on its type execute a controlling action through the final control element which is
often a pneumatically or electrically controlled valve. The control hardware is either analog or
digital. The analog system may be pneumatically operated using instrument air or electrically
through wire.
13.1. Different types of controllers
13.1.1 P controller
The proportional controller actuates the output proportional to the error. It gives considerable
offset but is simple and cheap and fast.
13.1.2. PI controller
It is a proportional plus reset controller. The integral action causes the controller output to
change as long as there is error in the output. Such a controller can eliminate small errors. It
produces zero offset but response in oscillating and sluggish
13.1.3. PID controller
It is a proportional plus reset plus rate controller. It anticipates what the error will be in future
and applies a control action proportional to the rate of change of error. However for response
with constant non-zero error it gives no control action. Also for small errors it may unnecessarily
bring about large control actions.

13.2. Control strategy for the Bioreactor
13.2.1. Temperature Control
Fermentation being an exothermic process, jacketed cooling water flow is used to maintain the
temperature of the fermentor at 37°C. This is done using a cascade control loop with the aid of
TIC01(primary controller) and FIC02(secondary controller ).PID controller is used since no
offset can be tolerated.
Measured variable: Temperature of Bioreactor, flow rate of cooling water in the jacket
Controlled variable: Temperature of Bioreactor
Manipulated variable: Flow rate of cooling water
13.2.2. Pressure Control
The control of pressure of the system is important from the safety point of view and to maintain
sterile conditions. During the fermentation process an internal pressure of about 1.2 bars is
desired to maintain sterile conditions. Although the pressure may go up to 1.3-1.4 bar during the
sterilization. Here a split range control loop is used controlling both the inlet air flow and the
vent flow rate through PIC01 to maintain a stipulated pressure inside the fermentor.PID
controller is used.
Measured variable: Pressure inside Bioreactor
Controlled variable: Pressure inside Bioreactor
Manipulated variable: Vent air flow rate, Inlet air flow rate
13.2.3. pH Control
The pH of the system needs to be maintained at about 6-7. As the fermentation proceeds the pH
of the broth continues to increase. The pH is maintained by the periodic addition of sulfuric acid
using a feed-back control strategy wherein the pH indicator pHI01 is connected to a controller
FIC01controlling the flow of acid into the system. Here a PI controller is used.

Measured variable: pH inside Bioreactor
Controlled variable: pH inside Bioreactor
Manipulated variable: Acid Flow rate
13.2.4. Control of foam formation
There may be a rise in the liquid level due to foam formation. This is controlled by the addition
of anti-foam when the liquid level (foam level) reaches a predetermined level. When the foam
reaches the probe tip, a current is passed through the circuit of the probe which gets completed,
with the foam acting as the electrolyte and the vessel as earth. Here again a feedback control
strategy is used wherein signals of level indicator are used to control the antifoamer flow rate. A
timer is also provided to ensure enough time for the antifoam to mix properly before more anti-
foam is added. A PI controller is used.
Measured variable: level inside Bioreactor
Controlled variable: level inside Bioreactor
Manipulated variable: Flow rate of antifoamer.

TT: Temperature transmitter
FT: Flow transmitter
LT: Level transmitter
PT: Pressure transmitter
PIC: Pressure indicator and controller
TIC: Temperature indicator and controller

14. HAZOP Studies
14. Hazard and Operability (HAZOP) Studies
The HAZOP study is a formal procedure to identify hazards and operational difficulties for a
given process and be prepared with corrective action for the same. The various terminologies
used in HAZOP analysis are:
Parameter: The important controlling factor.
Guidewords: The different guidewords used and their significance is as follows:
Table14.1. Guide words
Guide Words Meaning Comments
None The complete negation
of the intention
No part of the design intention is
achieved.
High, Too High Quantitative increase Applies to quantities such as flow rate
and to activities such as heating
Low, Too Low Quantitative decrease Applies to quantities such as flow rate
and to activities such as heating
Possible causes: Probable reasons for increase/decrease of parameter.
Possible consequences: Probable repercussions of increase/decrease of parameter.
Actions: Immediate activity to be performed to bring back normal functioning.
Safeguards: What should be done to prevent operating problems in the future .

14. HAZOP Studies
The table shown below presents HAZOP study done for the Bioreactor (B003):
Table 14. 2. HAZOP of B003
Parameter Guidewords Possible causes Possible
consequences
Actions Safeguards
Cooling
water flow
rate
Too high 1. Failure of
thermocouple.
2. Failure of
control valve.
3. Failure of
controller.
Decrease in
temperature - Not
optimum for
microbes
1. Manually
decrease flow rate
2. Increase
cooling water
temperature
1. Install
extra valve
(Bypass)
2. Install
alarm
Too low 1. Failure of
thermocouple.
2. Failure of
control valve.
3. Failure of
controller.
Increase in
temperature - poor
Yield
1. Manually
increase flow rate
2. Reduce
cooling water
temperature
1. Install
extra valve
(Bypass)
2. Install
alarm
Cooling
water
temperature
Too high Failure of
refrigeration
system
Increase in
temperature- poor
Yield
Increase flow rate 1. Install
cascade
control
2. Install
Alarms
Too low Setting error in
refrigeration
system
Decrease in
temperature-Not
optimum for
microbes
Decrease flow
rate
1. Install
cascade
control
2. Install
Alarms

14. HAZOP Studies
Impeller
Speed
Too high Operator or
mechanical
error
High Shear-
destruction of
microbes.
Manually reduce
impeller speed
Install alarm
Too low Operator or
mechanical
error
Poor distribution of
air.
Manually
increase impeller
speed
Install alarm
None Power failure or
failure of
impeller
No distribution of air
and no suspension of
microbes.
Switch on backup
power supply.
1. Install
generator
2. Install
alarm
Internal
pressure
Too high 1.Failure of
pressure valve
2. Failure of
pressure
controller
Explosion 1. Open pressure
relief valve
manually
2. Shut-off air
flow.
1. Install a
critical
alarm
2. Install
disc valve
that
automaticall
y releases
pressure.
Too Low Vent open /
leakage in valve
Contamination Seal valve. 1. Install an
extra valve
2. Regularly
carry out
maintenance
of valve.

15.Batch Scheduling
15.Batch Scheduling
Proper batch scheduling is important for achieving optimal productivity.Bioreactor is carried out
in fed- batch mode. Using the unsteady state equations,the time required for various activities is
found as follows
For sterilization of Bioreactor B002
Heat up time =36 min. Hold time = 45 min. Cool down time =10 min .
Table 15.1. Batch Scheduling
Activity Time(hrs)
Initial Down time for
Transformation/
Preparation
5
B001
Fermentation
Down time
TOTAL
48
2
50
Activity Time(hrs)
BOO2 (50L)
Fermentation
Down time
TOTAL
60
5.75(considered for lag
phase, preparation etc)
65.755
B003
Heating
Hold time
0.6
0.75

15.Batch Scheduling
Cooling
Fermentation
Emptying
TOTAL
0.1666
143.69
1.45
146.66
Centrifuge C001 1
F001 Affinity
Chromatography
0.5
F002 Ion exchange
Chromatography
0.5
F003 VRF 4
F004 UF 4
F005 DF 4
FC001 Formulation
column
1
The total time taken for the first batch is 282.41hrs.(11.76 days).Thereafter all the batches take
282.41hrs

16.MOC Selection
16. MOC Selection
For efficient working of the process, the stability of equipment is very important. This stability to
a large extent depends on its material of construction. The bioreactor vessel is made of steel,
metal or their combination. The relation between the height H and diameter D of the bioreactor is
within 1.5-2.5, There are high requirements for the reactor vessel materials to prevent the
inhibition of the microorganism growth. The same applies also to any other part (sensors, pipes,
etc.), which are installed inside the bioreactor vessel. Few important characteristics to be
considered when selecting a material of construction are:
16.1. Mechanical properties
This includes parameters like tensile strength, stiffness elastic modulus (Young’s modulus),
toughness , hardness , fatigue resistance, creep resistance. The selected MOC needs to have good
mechanical properties under the process conditions. During sterilization all the equipments are
exposed to intense temperatures and pressure. Hence the material chosen should possess superior
mechanical properties.
16. 2. Corrosion resistance
This is an important characteristic to be considered while choosing an MOC for the considered
process. The presence of aeration coupled with agitation and tendency of reduction in pH during
the process, demand a material with high corrosion resistance. To improve corrosion resistance
the chromium content in the material needs to be above 12 %.
16.3. Contamination.
For any biological process, highly aseptic environment is required. In industries such as the food,
pharmaceutical, biochemical, and textile industries, the surface finish of the material is as
important as the choice of material, to avoid contamination.
16.4. Cost
The material cost plays an important role in project cost evaluation. Hence an optimal choice
should be made which justifies the trade off between the cost and quality of material chosen.

16.MOC Selection
Based on the above criteria, All the metal parts should be made from stainless steel. The most
widespread brand of the stainless steel applied in bioreactors is 316L. The letter L indicates that
this steel is with a low composition of carbon. The inner surface of the stainless steel bioreactor
should be polished to about a mirror surface quality to facilitate the washing and sterilization
process. Welding should be carried out in a fully inert gas medium. The inert gas should be
argon, which fully replaces the air. With time, the application of the welding technology not
corresponding to the requirements can cause the corrosion of the welds. The MOC selected for
all other equipments in the plant is stainless steel.

17. Plant Layout
17. Plant Layout:
The general manufacturing site layout is as shown below:
Fire Station
Time
Keeping
Office
Securit
y
Office
Green Belt
Administration
Building
Canteen
Rest Room
Securit
y
Office
Records
Keeping
Workshop
And
Maintenance
Storage
Tanks
Medical
Facilities
Control
Room
Store
Room
MAIN PLANT AREA
Reserved Space
For future growth
10
Water
Chilling
Plant
Parking
Green Belt
ETP
ETP
Steam
Generation

17. Plant Layout
The equipment layout in the plant is as follows:
B003
S002 S004S003S001
B001 B002
S0001
S005
C001
F001F002F003F004FC001
V001 PRODUCT
STORAGE
S006

18.Financial Analysis
18. Financial Analysis
18.1. Project cost estimation.
For estimating the Project Cost (Fixed Capital Investment), we first estimate the Cost of equipment
(Delivered), The table below displays the key economic evaluation figures for the case of Rituximab.
18.1.1. Operating cost Breakdown
The sizing of all the equipments is shown in Chapter 11 Equipment Design and MOC selection is
discussed in chapter 12
Costs of equipments (plant and machinery) can be deduced as a direct function of the size of that
equipment and its material of construction, which are obtained from formulae, cost – capacity
correlations, graphs, etc.
In cases where all data pertaining to the equipment isn’t available, the following power – law model is
used to deduce the cost of the equipment.
n= 0.68: For general equipment.
0.63: For Fluid handling equipment.
0.63: For vessels, storage units etc.
Also, to take into consideration the time face the CE plant cost index needs to be taken.
Table 18.01Costs of various materials
Sr. Material Cost (Rs./kg)
No.
(including fabrication)
1. Carbon Steel 150
3. SS-316L 550

18.1.11 Gross Cost of installed equipment
Table 18.11: Gross cost of installment
Equipment Installmnent
Cost (Rs)
2.2 lit Roller Bottle 1800
DFT DEF cartridege 300000
225 ml T flsk 1200
DFT membrane 48000
100 lit cell bag 1260
Viral exclusion membrane 1602720
20 lit cel bag 24000
Protin A column 6480000
Ion exchange column 7200000
B001 1000000
B002 1800000
B003 1500000
C001 300000
F001 400000
F002 400000
F003 400000
F004 1200000
FC001 500000
V001 1000000
Freeze 1200000
Storage tank 500000
Compressor 1500000
Pump

The gross cost of installed equipment = Rs. 273.5898 Lakhs.
18.2 Estimation of plant and machinery cost
18.2.1 Equipment cost for Outside Battery Limit (OSBL) facilities
The OSBL facilities required are:
 Cooling unit for Dynalene MS 2 

 Storage facility for raw materials and products 

 Cooling Tower 
The Battery Limit plant has few pieces of equipment. Therefore, it would be reasonable to assume
that the cost of the OSBL facilities would be a fraction of the Battery Limit plant. We assume that it
is 30 % the cost of battery-limit equipment. In a similar way the piping, instrumentation, electrical,
installation and other components of plant and machinery costs are estimated.
18.2.2 Plant and Machinery cost
Table18.12: Breakup of OSBL
Component
% of
Equipment
cost
Cost
(lakhs)
Equipment 273.5898
Piping 20 54.71796
Instrumentation 20 54.71796
Electricity 15 41.03847
OSBL 30 82.07694
Total A 506.1411
Spare 5 % 0f A 25.30706
Subtotal B 531.4482
Packaging and forwarding 3 8.207694
Transportation 3 8.207694
Insurance 1 2.735898
Installation 15 41.03847
insulation and painting 2 5.471796
VAT 10 27.35898
Octrai 3 8.207694
Excise 15 41.03847
GRAND TOTAL plant and machine( Lakh) 673.7149

18.2.3 Estimation of total project cost
The project can be estimated based on rule of thumb by breaking up the project cost components as the
percentage of overall project cost. (Mahajani & Mokashi, 2005).
Table 18.13 Breakup of Total Capital Cost
Component % Cost
Land and site development 10 134.743
Building and civil work 8 107.794
Plant and machinary cost 50 673.715
Khow how and engineering fees 8 107.794
Miscellaneous Fixed Assets (MFA) 3 40.4229
Contingency 5 67.3715
pre operative expenses 13 175.166
Preliminary & Capital issue related
Expenses
3 40.4229
Total Project Cost (Lac) 100 1347.43
Total Project Cost = Rs 1347.43Lakh
= Rs 13.47Crores
Note: Price of Land is found from G.I.D.C website
Land and site development cost 134.7429765 Lakh
GIDC Rate 1500 Rs/m2
Area of land 8982.8651 m2
Area of land 2.201682623 Acres

20. Estimation of Working Capital
Working capital is the capital required to make the project work perform. The estimates are based on
the guideline by Mahajani & Mokashi (2005). The following are the components of working capital.
20.1 Raw Materials
We generally keep an inventory of 1 batch as storage of Raw Materials as work in progress. Steam is
also generated in plant from waste heat boiler and air is compressed whenever required so no need to
store it. The inventory, therefore, is not applicable for raw materials.
20.2 Product and Utility in stock
The amount is estimated at the cost of production.
Table 20.1 Product Streams as WIP and Utility costs for 1 batch
Product
No. of
days Amount(kg) Cost(Rs)/Kg
Total
Cost(Rs)
Product 15 0.5 12000000 6000000
0
Utilities 15 5 % of total 315789.4737
TOTAL PRODUCT COST ( Rs) 6315789.474
20.3 Maintenance Spares
Calculating the plant and machinery item of project cost for 1 month.
The total plant and machinery cost of the project = 673.7149 lakhs.
A provision is made for maintenance spares as 0.1% of the plant and machinery item of the project
cost for one month.
Thus, maintenance spare inventory cost = Rs. 0.67374883 lakhs.
20.4 Other fixed costs
These are estimated as 10% of the product inventory cost.
Thus, other fixed expenses = 10% of (644) lakhs.
= Rs. 6.31578947 lakhs.

Table 20.2 Total working capital break-up
Parameter Cost(lakh)
Raw material inventory cost 49.232858
Product inventory cost 63.1578947
Thus, maintenance spare inventory cost 0.67371488
Other fixed cost 6.31578947
TWC (Lac) 119.380257
20.5 Working capital source
Following table shows source of total working capital.
Table 20.3 Source of working capital
Component Contribution Cost in lakh
Borrowed WC 75 % TWC 89.53519
Margin money 25% TWC 29.84506427
Total Working Capital ( Lac) 119.3802571

19. Total Cost of Production
After finding the Total Capital Investment, we need to estimate the cost of production of
each kg of the product. This has significant impact on selling price and hence the
profitability of the project. Following are the main components of cost of production.
It is assumed that the plant runs continuously for 300 days in a year.
19.1 Raw Material Cost
Table 19.1: Cost of Raw Materials
Raw material Batch req
(kg)
Rate(Rs/kg) Annual
req(kg)
Cost (Rs)
Inoc. Media Sol 129 368 2580 949440
serum free media 246 18000 4920 88560000
Air 10525 0 210500 0
H3PO4 6275 8.5 125500 1066750
NaOH 5892 15 117840 1767600
Pro. A reg buff 3174 10 63480 634800
Pro.A elution 5317 9.2 106340 978328
Pro A equil 11562 9.2 231240 2127408
ION eq buff 1730 11.2 34600 387520
ION wash buff 1732 22 34640 762080
IOX el buff 97 18.5 1940 35890
Nacl(1M) 1065 22 21300 468600
amm. Sulfate 74 480 1480 710400
polysorbate80 2 110 40 4400
sodium citrt dihydrate 0.25 2500 5 12500
Total raw material cost 98465716
19.2 Utilities Cost
Table 19.2: Cost of Utilities
Utilities requirement
Annual
requirement Rate (Rs./ unit) Cost per annum
Cooling water 50000 Kg Rs. 15/m3 750
Electricity 58000 KW.hr Rs.8 /KW.hr 464000
Steam 80000 kg Rs.1.4/kg 112000
Total Utilities cost per annum (Rs.) 576750

Thus, Variable Cost = Raw Material Cost + Utility Cost
= 990.42466 lakhs
19.3 Indirect cost
Table 19:3 Indirect Product Cost
Component Contribution Total Cost (Rs. In
lakhs)
Operating labour cost (OLC) 10 % of project cost 134.7429765
Repairs and maintainance 5 % of project cost 67.37148825
Supervision 15 % of OLC 20.21144648
Labour 15 % of OLC 20.21144648
Supplier 15 % of M & R 10.10572324
Indirect product cost (Rs. In lakh) 252.6430809
19.4 Fixed Charges
Table 19:4 Fixed Charges
Component Contribution
Total Cost (Rs. In
lakhs)
Local tax
4 % of project
cost 53.8971906
Insurance
1 % of project
cost 13.47429765
Total fixed charges ( Rs. In lakh) 67.37148825
19.5 Plant Overheads
Plant overheads = 40 % (OLC + Supervision + Repairs and Maintenance Cost)
= Rs. 88.930 lakh
Total plant Overheads (Rs. In lakh) = 88.930 lakh

19.6 General Expenses
Table 19:5 General Expenses
Component Contribution Total Cost (Rs. In
lakhs)
Administration cost 25 % of OLC 33.68574413
Distribution and selling 0.2 % of project
cost
2.69485953
Generl expences ( Rs. In lakh) 36.38060366
19.7 Salary & Wages
Salary and Wages = 9 % of variable cost
= 0.09 x (990.42)
Salary and Wages = Rs. In laks 1.0903/-
Therefore fixed cost = Total indirect cost + Fixed charges + Plant Overheads + General
Expenses + Salary and Wages
= Rs. 446.415 lakh
Fixed cost = Rs. 446.415 lakh.
19.8 Cost of Production
Cost of production = Variable cost + Fixed cost
Total cost of production =Rs1436.840592 in lakh.
Now,
We manufacture 10 kg of RITUXIMAB.
Cost of production = Rs. 14368405.92=14370000/kg
19.9 Comments on Economic Feasibility
Following is the production capacity of products and revenue generated from selling product:
Products Rate ($/kg) Annual Capacity(Kg) Rate(Rs)/Kg
total
Cost(Rs)
Product 300 10 18000000 180000000

Weighted average selling price of product = Rs. 1800000/kg
Hence there is an earning of Rs. 3630000/kg and therefore Rs36300000 lakh / annum.
Since the project is economically feasible. We can proceed to estimating the working capital and
do further financial analysis of the project.
19.10 Summary
Table 19.6 Summary of Production Cost
Sr. No. Type Component Cost( Rs.
in Lakh)
1 Variable Raw material 984.65
2 Utilities 5.76
Variable Cost of Production 990.42
3 Fixed Indirect cost 252.64
4 Total fixed charges 67.37
5 Plant overheads 88.93
6 General expenses 36.38
7 Salary and wages 10.90
Fixed cost of Production 446.40
Total cost of Production 1437.00

Working capital is the capital required to make the project work perform. The estimates are
based on the guideline by Mahajani & Mokashi (2005). The following are the components of
working capital.
20.1 Raw Materials
We generally keep an inventory of 1 batch as storage of Raw Materials as work in progress.
Steam is also generated in plant from waste heat boiler and air is compressed whenever required
so no need to store it. The inventory, therefore, is not applicable for raw materials.
20.2 Product and Utility in stock
The amount is estimated at the cost of production.
Table 20.1 Product Streams as WIP and Utility costs for 1 batch
Product
No. of
days Amount(kg) Cost(Rs)/Kg
Total
Cost(Rs)
Product 15 0.5 12000000 6000000
0
Utilities 15 5 % of total 315789.4737
TOTAL PRODUCT COST ( Rs) 6315789.474
20.3 Maintenance Spares
Calculating the plant and machinery item of project cost for 1 month.
The total plant and machinery cost of the project = 673.7149 lakhs.
A provision is made for maintenance spares as 0.1% of the plant and machinery item of the
project cost for one month.
Thus, maintenance spare inventory cost = Rs. 0.67374883 lakhs.
20.4 Other fixed costs
These are estimated as 10% of the product inventory cost.
Thus, other fixed expenses = 10% of (644) lakhs.
= Rs. 6.31578947 lakhs.

Table 20.2 Total working capital break-up
Parameter Cost(lakh)
Raw material inventory cost 49.232858
Product inventory cost 63.1578947
Thus, maintenance spare inventory cost 0.67371488
Other fixed cost 6.31578947
TWC (Lac) 119.380257
20.5 Working capital source
Following table shows source of total working capital.
Table 20.3 Source of working capital
Component Contribution Cost in lakh
Borrowed WC 75 % TWC 89.53519
Margin money 25% TWC 29.84506427
Total Working Capital ( Lac) 119.3802571

21. Estimation of Financial Expenses
21. Financial Analysis
Financial Analysis involves estimation of financial expenses, depreciation values, key
indicative ratios and the breakup of working capital as a function of time, all of which helps
plan the execution and operations of the project.
Table 21.1 Project Financing Components
Total fixed capital investment 1347.43 Lac
Debt : Equity 1.5:1
Term Loan 808.4579 lac is @12 % pa
Equity participation 538.9719 lac
Promotors contribution 274.8757 lac 51%
Organizational participation 80.84579 lac 15%
Public issue 183.2504 lac 34%
Borrowed working capital 89.53519 lac is @ 16 % installment for 4
years
21.1 Estimation of Financial Expenses
Term loan repayment period = 10 years; yearly in equal half annual installments
Moratorium period = 2 years; after which equal installments are paid
Working Capital Repayment Period = 4 year; annually in equal half annual installments
Interest is found by computing simple interest for that period.

Table 21.2 Term Loan Scheduling
Table 21.3 Borrowed Working Capital Repayment Schedule
Installment
Principle
remaining Intrest payble
Principle
paid
Installment
paid
Principle at
year end
Rs in Lac Rs in Lac Rs in Lac Rs in Lac Rs in Lac
1 89.54 7.16 8.42 15.58 81.12
2 81.12 6.49 9.09 15.58 72.03
3 72.03 5.76 9.82 15.58 62.21
4 62.21 4.98 10.60 15.58 51.60
5 51.60 4.13 11.45 15.58 40.15
6 40.15 3.21 12.37 15.58 27.78
7 27.78 2.22 13.36 15.58 14.43
8 14.43 1.15 14.43 15.58 0.00
Installment
Principle
remaining Interest payable
Principle
paid
Installment
paid
Principle at
term end
Rs in Lac Rs in Lac Rs in Lac Rs in Lac Rs in Lac
1 808.46 48.51 0.00 48.51 808.46
2 808.46 48.51 0.00 48.51 808.46
3 808.46 48.51 0.00 48.51 808.46
4 808.46 48.51 0.00 48.51 808.46
5 808.46 48.51 31.49 80.00 776.97
6 776.97 46.62 33.38 80.00 743.59
7 743.59 44.62 35.38 80.00 708.20
8 708.20 42.49 37.51 80.00 670.70
9 670.70 40.24 39.76 80.00 630.94
10 630.94 37.86 42.14 80.00 588.80
11 588.80 35.33 44.67 80.00 544.13
12 544.13 32.65 47.35 80.00 496.78
13 496.78 29.81 50.19 80.00 446.58
14 446.58 26.79 53.20 80.00 393.38
15 393.38 23.60 56.40 80.00 336.98
16 336.98 20.22 59.78 80.00 277.20
17 277.20 16.63 63.37 80.00 213.84
18 213.84 12.83 67.17 80.00 146.67
19 146.67 8.80 71.20 80.00 75.47
20 75.47 4.53 75.47 80.00 0.00

21.2 Depreciation Schedule
To estimate depreciation, the preoperative expenses and contingencies are distributed among
the other project cost components, in their corresponding proportions. Following table
contains the depreciation rates are used for the estimation of the working results. The rates of
depreciation are fixed by the Income Tax Act and can be revised during the budget.
Table 21.4 Depreciation Rates
Depreciation rate
Cost Component Value Depreciation rate
Rs in Lac SLM (%) WDV (%)
Land and site development ( L&D) 134.74
Building and Construction ( B& C) 107.79 3.34 10.00
Plant and Machinery (P & M) 673.71 10.34 25.00
Misc.fixed assets (MFA) 40.42 5.00 20.00
SLM: Straight line method.
WDV: Written Down Value Method.
In case of straight line method, the asset depreciates at a constant rate and the value after n
years is given by (Mahajani & Mokashi 2005):
Va  Vo  DSLM Vo  n Equation 23.1
In case of written down value method (also known as accelerated depreciation method), the
value of asset at the end of any year n is given by:
Va  Vo  1  DWDV n
Equation 23.2
In the above equations:
Va = Value of asset at the end of year n,
Vo = original value,
DSLM = Depreciation rate by straight line method, %.
DWDV = Depreciation rate by written down value method, %.

The depreciation schedule is as follows:
Table 21.5 Depreciation Schedule
Year MFA B&C P&M Total
SLM WDV SLM WDV SLM WDV SLM WDV
1 2.02 8.09 3.60 10.78 69.66 168.43 75.28 187.30
2 2.02 6.47 3.6 9.70 69.66 126.32 75.28 142.49
3 2.02 5.17 3.6 8.73 69.66 94.74 75.28 108.65
4 2.02 4.14 3.6 7.86 69.66 71.06 75.28 83.05
5 2.02 3.31 3.6 7.07 69.66 53.29 75.28 63.67
6 2.02 2.65 3.6 6.36 69.66 39.97 75.28 48.98
7 2.02 2.12 3.6 5.73 69.66 29.98 75.28 37.82
8 2.02 1.70 3.6 5.16 69.66 22.48 75.28 29.33
9 2.02 1.36 3.6 4.64 69.66 16.86 75.28 22.86
10 2.02 1.09 3.6 4.18 69.66 12.65 75.28 17.91
21.3 Sales Realisation
Table 21.6 Sales Realisation
Product Annual
production
Selling Price (
Rs/kg)
Sales(
Lac/Year)
Rituximab 10 18000000 1800
21.4 Break-Even Analysis
The following assumptions are made while carrying out the break-even analysis (Mahajani &
Mokashi, 2005):
 All expenses are bifurcated into fixed and variable costs. 
 The market conditions are ideal. Whatever is produced is sold immediately. 
 The selling expenses are fixed as some % of sales. 
 The following annualized (fixed) costs are not dependant on capacity utilization, namely,
interest, charges on term loan, depreciation. 

The equation used to estimate the break even capacity is: 
S * X= V * X + F Equation 23.3

where, X = Break-even capacity
S = Price of one ton of product
V = Variable cost of production per ton
F = Fixed cost of production = Interest on Term Loan + All Overhead costs
Therefore, X = F/(S-V) Equation 23.4
The fixed cost of production includes the overheads, administrative expenses, average
financial expenses and depreciation (SLM). Substituting the required values,
F = 326.08 lakh/ annum
V =
Similarly, the sale price for products in the above proportion can be calculated as:
S= n
X= 0.004027ton/ annum
X =40.28 %.

21.5 Ratio Analysis
21.5.1 Performance Ratios
A. Payback Period
Assuming 100 % capacity utilization
Table 21.7 Payback Period
Gross cost of production 143700000 Rs.
annual sales 180000000 Rs.
Gross profit 36300000 Rs.
Total capital investment 130222198.1 Rs.
Payback period
3.587388378 Years
3 Year and 7months
B. Return on Investment (ROI):
Assuming 100% capacity utilization, ROI is given by,
Gross Profit * 100 Equation 21.5
Total Capital Investment
Return on Investment = 27.87 %65.78%
C. Profit Margin:
Assuming 100% capacity utilization,
Profit margin = ( Gross profit / Sales) * 100 Equation 21.6
Profit margin = 20.16 %
D. Net Assets Turnover:
Net assets turnover = (Sales / Project cost) Equation 21.7
Net assets turnover = 1.33

21.5.2 Financial Ratios
A. D:E Ratio
Debt : Equity = 1.5:1
B. Interest Cover:
100% capacity utilization is assumed and interest cover is calculated for the first year.
Interest Cover = Gross profit / Interest Equation 21.8
Interest Cover = 3.26
21.6. Estimates of Working Results
The projected working results for 10 years are presented in tabulated form. A dividend of
20% is assumed from the first year. Following are the terms involved in this evaluation:
 Gross Profit = Sales – Gross Cost of Production
 Operating Profit = Gross Profit – Financial Expenses – SLM Depreciation
 Taxable Profit = Gross Profit – Financial Expenses – WDV Depreciation – Loss from
previous year (if any)
 Corporate Tax = 30% of Taxable Profit
 Profit after Tax = Gross Profit – Financial Expenses – Corporate Tax
 Profit for Dividend = Profit after Tax – SLM Depreciation
 Dividend = 20% of Profit for Dividend
 Profit after Dividend = Profit for Dividend – Dividend
 Net Cash Accruals = Profit after Dividend + Depreciation(SLM)

Table 21.8 Estimate of working capital for years 1-5(All figures in
Rs. (Lakhs), except capacity and utilization)
Year 1.0000 2.0000 3.0000 4.0000 5.0000
Capacity utilisation 70.0000 70.0000 80.0000 80.0000 90.0000
% capacity 0.0070 0.0070 0.0080 0.0080 0.0090
Sales 1260.0000 1260.0000 1440.0000 1440.0000 1620.0000
Gross cost of production 1005.9000 1005.9000 1149.6000 1149.6000 1293.3000
Gross profit 254.1000 254.1000 290.4000 290.4000 326.7000
Financial expenses 97.0149 97.0149 95.1255 87.1073 78.0981
Depriciation ( SLM) 75.2836 75.2836 75.2836 75.2836 75.2836
Depriciation ( WDV) 120.2100 115.0700 108.6452 83.0523 63.6748
Operating profit 81.8015 81.8015 119.9909 128.0091 173.3183
Taxable profit 36.8751 42.0151 86.6293 120.2404 184.9271
Corporate profit 11.0625 12.6045 25.9888 36.0721 55.4781
Profit after tax 146.0225 144.4805 169.2857 167.2206 193.1238
Profit for devident 70.7389 69.1969 94.0021 91.9370 117.8402
devidend 14.1478 13.8394 18.8004 18.3874 23.5680
Profit after devidend 56.5912 55.3576 75.2017 73.5496 94.2721
Net cash accural 131.8748 130.6412 150.4853 148.8332 169.5557
Table 21.9 Estimate of working capital for years 6-10 (All figures
in Rs. (Lakhs), except capacity and utilization)
Year 6.0000 7.0000 8.0000 9.0000 10.0000
Capacity utilisation 90.0000 95.0000 95.0000 100.0000 100.0000
% capacity 0.0090 0.0095 0.0095 0.0100 0.0100
Sales 1620.0000 1710.0000 1710.0000 1800.0000 1800.0000
Gross cost of production 1293.3000 1365.1500 1365.1500 1437.0000 1437.0000
Gross profit 326.7000 344.8500 344.8500 363.0000 363.0000
Financial expenses 67.9754 56.6015 43.8218 29.4625 13.3284
Depriciation ( SLM) 75.2836 75.2836 75.2836 75.2836 75.2836
Depriciation ( WDV) 48.9825 37.8241 29.3333 22.8580 17.9073
Operating profit 183.4410 212.9649 225.7446 258.2539 274.3880
Taxable profit 209.7421 250.4245 271.6950 310.6795 331.7643
Corporate profit 62.9226 75.1273 81.5085 93.2038 99.5293
Profit after tax 195.8020 213.1212 219.5197 240.3337 250.1424
Profit for devident 120.5184 137.8376 144.2362 165.0501 174.8588
Dividend 24.1037 27.5675 28.8472 33.0100 34.9718
Profit after dividend 96.4147 110.2701 115.3889 132.0401 139.8870
Net cash accrual 171.6983 185.5537 190.6725 207.3237 215.1706

21.7 Discounted Profit Flow Analysis
A. Weighted Average Cost of Capital
The weighted average cost of capital is given by:
WACC
Di
T
Er
E Equation 21.9
D  E
For our project, D:E = 1.5:1
iT= 0.12
rE= 0.30 (assume).
We have WACC = 0.192 = 19.2%
B. Hurdle Rate of Return (K):
K= 22.5% (> WACC)
C. Present Value of Profit (PV)
The present value of profit is given by:
∑
Equation 21.10
where,
Pn denotes the Gross Profit Projection for the nth year.
Substituting the values, we have,
PV = Rs. 1670.986746 lakhs.
D. Present Value of Investment
The total capital investment is Rs. 7493 lakhs. Out of these, assume that Rs. 6000 lakhs are
spent in the first year of investment and the rest are spent in the second year. It takes two
years for the construction of project.

Present Value of Investment = Io  (1  K ) I1I2 

= Rs.1414.92975akhs.
E. Net Present value of Profit
The net present value of profit is given by:
∑
Equation 21.11
= 256.056 Lakhs
The project thus seems attractive.
F. Profitability Index:
The Profitability Index is given by:
∑
Equation 21.12
PI = 1.18
G. Internal Rate of Return:
The Internal rate of return (IRR) is the value at which the NPV is zero. It is given by:
∑
Equation 21.13
IRR = 52.2%
As IRR is greater than WACC, project is attractive.

22. Storage, Utilities and Effluent treatment
22. Storage, Utilities and effluent treatment
22.1. Storage
The entire process demands high level of monosepsis. Hence both the raw materials and the
products need to be stored in extremely sterile environment. The raw materials are mostly
biological grade materials and are stable at room temperature. The product cannot be exposed to
high temperature. In order to retain its activity for a long time it is stored at -25°C.
22.2. Utilities
The utilities required in the plant are 4 bar saturated steam ,cooling water and electricity
Amount of steam required for sterilization of Bioreactor =2280+11+5.5≈3000kg.Besides steam
is also required to sterilize the pipelines and other equipment. Hence total steam requirements is
4000kg
Cooling water is consumed during sterilization and the fermentation process. Total cooling water
required =214788+520+1042+5.5×3600×85=1900tonnes≈1900 m3
.Besides water forms a major
portion on the fermentation medium too.
Cooling water is re-circulated and so for annual requirement we only need to take consideration
losses of evaporation and other losses. Let, cycle time of 1 day. So water required per batch is
126.66 TPB.
Water losses = M = 1.73 ×0.001 ×∆T × Vc × (Kc/Kc-1)
Water losses per batch are = 2.5 Ton
Annual fresh water required is 50 Ton

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