Beruflich Dokumente
Kultur Dokumente
BY
ANIL V VIBHUTE
INSTITUTE OF CHEMICAL TECHNOLOGY
MATUNGA, MUMBAI - 400019
2013-2014
YG
INDEX
1 Introduction 1
2 Executive summary 5
3. Process selection 7
4. Process Description 9
5.1 Kinetics 17
5.2 Thermodynamics 19
6 Block Diagram 20
7 Site Selection 21
8 Material Balance 25
9 Energy balance 34
12 Equipment Sizing 55
15 Batch Scheduling 69
16 MOC Selection 71
17 Plant Layout 73
18 Financial Analysis 75
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB
INDEX
24 Conclusions 100
25 References 101
26 Appendix A 103
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB
1. Introduction
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.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 1
1. Introduction
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
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)
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
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 2
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
6 Appearance
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 3
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
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 4
2. Executive Summary
2. Executive Summary
Product: RITUXIMAB
Annual
Raw material Batch req ( kg) Rate (Rs/kg) cost(Rs)
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 5
2. Executive Summary
Sales:
Financing:
Project Evaluation:
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 5
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:
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.
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.
4) Most of the raw material required are easily available from Mumbai.
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:
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
4.Process description.
Batch reactor
Reaction-1 Reaction-3
Filtration
Y-3
a) Materials
Powder with high glucose with L-glutamine with pyridoxine hydrochloride with 110 mg/L
sodium pyruvate without sodium bicarbonate
▪ 0.22 µm vacuum filter (vaccucap 90): Gelman science #pn 4622-filterling capacity up to 10 L
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 10
4.Process description.
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.
- 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).
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.
(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
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 11
4.Process description.
c) Preparation of DMEM
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,
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
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.
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,
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 12
4.Process description.
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.
This media is suitable for non-transfected CHO K1, or transiently transfected CHO cells which
do not have any antibiotic resistance.
1. 430 mL of DMEM
3. 5 mL of Pen/Strep
5. 10 mL of L-glutamine
This media is suitable for CHO K1 cells which stably express fluorescent proteins and are
resistant to geneticin.
2.50 mL of FBS
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 13
4.Process description.
3.5 mL of Pen/Strep
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.
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:
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.
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
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.
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
5. Kinetics and thermodynamics of the 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:
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 17
5. 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.. Hence proper conditions and control is required for maximum
yield. Below the growth trend and substrate consumption for the CHO is given.
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
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 18
5. Kinetics and thermodynamics of the process
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.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 19
6. Process Block Diagram
Nutrient
Medium Inoculum
Preparation
Air VR filtration
Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 20
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
7.1.3. 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.
Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 21
7. Site Selection
7.1.6 .Power:
Regular and uninterrupted power supply at conceded rates is an essential factor to run the project
smoothly.
7.2.2 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.
Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 22
7. Site Selection
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.
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.
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
Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 23
7. 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
Manufacture of 100000, 10ml fill vials per annum at 10 mg/ml Strength Of RITUXIMAB Page 24
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.
Total batches = 20
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 25
8. Material Balance
Volume = 25 Liters
1
B001 3
2
2
Volume = 50 Liters
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 26
8. Material Balance
3
B002 5
4
2
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.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 27
8. Material Balance
5
B003 7
6
2
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 28
8. Material Balance
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.
7 C001 9
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).
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 29
8. Material Balance
F001 11
9
10
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.
11 F002 13
12
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 30
8. Material Balance
13 F003 15
14
15 F004 17
16
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 31
8. Material Balance
17
3 FC001
18
17 10 P002
12
13
10 P002 13
21
12
19
10 P002 13
12
20
10 P002 13
12
input(gm) output(Kg)(20)
Polysorbate-80(17) 37.97 0
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 32
8. Material Balance
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 33
9. Energy balance
9. Energy balance
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
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
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.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 34
9. Energy balance
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
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 35
10. Equipment Design
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 36
10. Equipment Design
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
( )
6.515W
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 37
10. Equipment Design
( )
10.1.5.Sparger design
Gas hold up in the system (Handbook of chemical engineering calculations, Pg no. 575)
( )
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 38
10. Equipment Design
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).
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 39
10. Equipment Design
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 40
10. Equipment Design
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 41
10. Equipment Design
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.
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.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 42
10. Equipment Design
Continuous
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 43
10. Equipment Design
25
96
23
94
21
19 92
17 90
15 88
0 1 2 3 4 5 6
Diafiltration Volume
-UNIT NUMBER:
Stainless stain housing
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 44
10. Equipment Design
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 45
11. Mechanical design Of Bioreactor
H =0.41m D= 0.39m
= 2.23 mm
P.Rc.W
th + C
2.f.J
1 Rc
[3 ]
4 R1
= 1.77
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 46
11. Mechanical design Of Bioreactor
th = 0.41+2 = 2.41 mm
At the bottom:
Here J = 1
So we use 4 mm thickness
Go Ya mp
Gi Ya mp p
Go
1.0013
Gi
Go = 400.54mm
A flat asbestos gasket of 390 mm internal diameter and 400 mm external diameter is used.
Under atmospheric conditions bolt load due to gasket reaction is given by:
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 47
11. Mechanical design Of Bioreactor
Wm1 = b G Ya
Wm2 = .(2b).G.m.P .G 2 .P
4
= 8.42x 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
Bolt area = d 2 * 78
4
Pitch circle diameter (P.C.D) = Outside diameter of gasket + 2 diameter of bolt + 12mm
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 48
11. Mechanical design Of Bioreactor
P
tf G
Kf
1
where K
1.5Wm h g
0.3
HG
= (PCD - G)/2
= 34.5 mm
= G2P
4
= 0.6 x 106 N
K = 2.69
tf = 44.6 mm
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 49
11. Mechanical design Of Bioreactor
3) On shell:
P di
tn =
2fJ - P
=0.154*300/(2*1*140 - 0.154)
= 0.165 mm
A = d ts `
= 300 x 1.42
= 426 mm2
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 50
11. Mechanical design Of Bioreactor
Ah = d (ts – ts`)
Where
d = Diameter of nozzle
As = 1374 mm2
An = 2 x Ho x (tn - tn`)
Ho = 2.5 x tn
An = 29.175 mm2
Selection of support:
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 51
11. Mechanical design Of Bioreactor
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
Wmax L b 4
f h 0.7 4
b * l th b L
4
Where,
l= 75 mm
b=h=200 mm
L= 300 mm
Hence , th = 0.51mm
th = 2 mm
3Wmax l
tg
f h h 2COS
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 52
11. Mechanical design Of Bioreactor
Θ = 45 o
Hence tg = 1.22 mm
We take tg = 2 mm
= 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
Tm
Zp =
fs
d3
Zp =
16
(×d3)/16= 3.63cm3
d = diameter of shaft
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 53
11. Mechanical design Of Bioreactor
d = 2.65cm
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 54
12. Equipment Sizing
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 55
12. Equipment Sizing
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
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 56
12. Equipment Sizing
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 57
12. Equipment Sizing
Chemical Stability All commonly used buffers, nonionic detergents, 1 M NaOH, 6 M guanidine
hydrochloride
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 58
12. Equipment Sizing
Chemical Stability All commonly used buffers, nonionic detergents, 1 M NaOH, 6 M guanidine
hydrochloride
Ligand Sulphopropyl
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 59
12. Equipment Sizing
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 60
12. Equipment Sizing
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
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 61
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.
13.1.1 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
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.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 62
13. Instrumentation and Process 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
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.
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.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 63
13. Instrumentation and Process control
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.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 64
13. Instrumentation and Process control
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 65
14. 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:
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
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 66
14. HAZOP Studies
The table shown below presents HAZOP study done for the Bioreactor (B003):
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 67
14. HAZOP Studies
Impeller Too high Operator or High Shear- Manually reduce Install alarm
Speed mechanical destruction of impeller speed
error microbes.
Too low Operator or Poor distribution of Manually Install alarm
mechanical air. increase impeller
error speed
None Power failure or No distribution of air Switch on backup 1. Install
failure of and no suspension of power supply. generator
impeller microbes. 2. Install
alarm
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 68
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
Heat up time =36 min. Hold time = 45 min. Cool down time =10 min .
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 5.75(considered for lag
phase, preparation etc)
TOTAL 65.755
B003
Heating 0.6
Hold time 0.75
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 69
15.Batch Scheduling
Cooling 0.1666
Fermentation 143.69
Emptying 1.45
TOTAL 146.66
Centrifuge C001 1
F001 Affinity 0.5
Chromatography
F002 Ion exchange 0.5
Chromatography
F003 VRF 4
F004 UF 4
F005 DF 4
FC001 Formulation 1
column
The total time taken for the first batch is 282.41hrs.(11.76 days).Thereafter all the batches take
282.41hrs
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 70
16.MOC Selection
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.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 71
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.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 72
17. Plant Layout
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
17. Plant Layout
B001 B002
B003 C001
S005
S0001
S006
V001 PRODUCT
STORAGE
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 74
18.Financial Analysis
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.
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.
Also, to take into consideration the time face the CE plant cost index needs to be taken.
3. SS-316L 550
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 75
18.Financial Analysis
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
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 76
18.Financial Analysis
VAT 10 27.35898
Octrai 3 8.207694
Excise 15 41.03847
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 77
18.Financial Analysis
= Rs 13.47Crores
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 78
18.Financial Analysis
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.
Table 20.1 Product Streams as WIP and Utility costs for 1 batch
No. of Total
Product days Amount(kg) Cost(Rs)/Kg Cost(Rs)
Product 15 0.5 12000000 6000000
0
Utilities 15 5 % of total 315789.4737
TOTAL PRODUCT COST ( Rs) 6315789.474
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.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 79
18.Financial Analysis
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 80
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
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 79
19. Total Cost of Production
= 990.42466 lakhs
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 80
19. Total Cost of Production
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
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 81
19. Total Cost of Production
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
2 Utilities 5.76
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 82
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.
Table 20.1 Product Streams as WIP and Utility costs for 1 batch
No. of Total
Product days Amount(kg) Cost(Rs)/Kg Cost(Rs)
Product 15 0.5 12000000 6000000
0
Utilities 15 5 % of total 315789.4737
TOTAL PRODUCT COST ( Rs) 6315789.474
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.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 83
20. Estimation of Working Capital
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 84
21. Estimation of Financial Expenses
Term loan repayment period = 10 years; yearly in equal half annual installments
Working Capital Repayment Period = 4 year; annually in equal half annual installments
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 85
21. Estimation of Financial Expenses
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 86
21. Estimation of Financial Expenses
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.
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):
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:
Vo = original value,
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 87
21. Estimation of Financial Expenses
S * X= V * X + F Equation 23.3
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 88
21. Estimation of Financial Expenses
The fixed cost of production includes the overheads, administrative expenses, average
financial expenses and depreciation (SLM). Substituting the required values,
V=
Similarly, the sale price for products in the above proportion can be calculated as:
S= n
X= 0.004027ton/ annum
X =40.28 %.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 89
21. Estimation of Financial Expenses
C. Profit Margin:
Assuming 100% capacity utilization,
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 90
21. Estimation of Financial Expenses
B. Interest Cover:
100% capacity utilization is assumed and interest cover is calculated for the first year.
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:
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 91
21. Estimation of Financial Expenses
Table 21.9 Estimate of working capital for years 6-10 (All figures
in Rs. (Lakhs), except capacity and utilization)
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 92
21. Estimation of Financial Expenses
iT= 0.12
where,
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.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 93
21. Estimation of Financial Expenses
∑
Equation 21.11
= 256.056 Lakhs
F. Profitability Index:
The Profitability Index is given by:
Equation 21.12
PI = 1.18
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%
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 94
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
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.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 95
22. Storage, Utilities and Effluent treatment
Utility Amount/batch
Steam 4000 kg
Water 1900 m3
Electricity 2900 kW.hr
Various solid, liquid and gas effluents are generated during the process. The basic aerobic
fermentation can be represented by the following equation
The fermentation process causes a lot of odor problems because of various volatile organic
compounds let out into the atmosphere during the process. In order to decrease the odor
intensity, the vent gases are passed through biofilters before letting them out to the atmosphere
Around 5 kg of sludge/batch is generated. This sludge can be reused, by direct land application
for soil conditioning and fertilization ,or by stabilization via chemical treatment with cement,
sodium silicate, lime etc for road subsoil additives, cement manufacturing etc.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 96
24. Conclusions
24. Conclusions
The technical and economic feasibility of putting up a 100000 vials of Rituximab plant in
India has been reviewed. On the basis of the calculations for the economic feasibility of the
process, total project cost comes out to be Rs. 1437 lakhs giving a return on investment (ROI) of
65.41 % (payback period of 43 months). The profitability index value of 1.18 (greater than 1)
shows the profitability of the project. Thus, the project on the basis of the above analysis
indefinitely profitable and economically feasible.
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. This even gives an opportunity of forward
integration of the plant to any of the products mentioned above. The location at Raigad,
Maharashtra ensures that raw material availability is never a problem as well as the export and
import is easy.
So, we can conclude that setting up this plant in India would lead to substantial profits.
However, it is to be noted that this feasibility study has been done considering some ideal
situations. Also, the project costs may vary depending process licensing fees, shipping of
proprietary equipment, etc. Some of the assumptions made are stability of raw material,
product prices and market demands, 100% product sale and negligible losses in material and
energy. It has been assumed that the plant functions to its full capacity from commencement
of production, which has contributed in reduction of the payback period. The time value of
money also has not been accounted for. To get clearer scenario, a detailed feasibility report
needs to be done. This report justifies the need to conduct a detailed feasibility report to come
up with more realistic figures.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of R ITUXIMAB Page 100
25.References
25. References
Diekmann SAD, 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, 2001: 103
Kelley B. Very large scale monoclonal antibody purification: The case for conventional unit
operations. Biotechnol Prog 2007; 23:995-1008
Perry R. H., Green D. W. and Malony J. O., “Perry’s Chemical Engineers Handbook”, 7th
Edition, McGraw-Hill, Sydney. (1999)
Sinott, R.K., “Coulson and Richardson’s Chemical Engineering Series: Chemical Engineering
Design”, Volume 6, 4th Edition, Butterworth-Heinemann (an imprint of Elsevier), New
Delhi. (2006)
Uhlig, H.H., “Corrosion Handbook”, John Wiley and Sons Inc., New York. (1948).
Walas, S.M., Fair R.J., Penny R.W. and Couper R.J., “Chemical Process Equipment: Selection
and Design”, Butterworth-Heinemann, Newton (USA). (1990)
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 101
25.References
Rao, C.S., “Environmental Pollution Control Engineering”, Wiley Eastern Ltd., New Delhi.
(1995)
Mahajani, V.V. and Mokashi, S.M., “Chemical Project Economics”, Maclmillan India Ltd., New
Delhi. (2005)
McCabe, W. L., Smith, J. C. and Harriot, P., “Unit Operations of Chemical Engineering”, 6th
edition, McGraw Hill Inc., New York. (2001)
Joshi, M. V. and Mahajani, V. V., “Process Equipment Design”, 3rd Edition, Macmillan India
Limited, India. (1996)
Richardson, J.F. with Backhurst, J.R. and Harker, J.H., “Coulson and Richardson’s Chemical
Engineering: Particle Technology and Separation Processes”, Volume 2, 5th Edition,
Butterworth-Heinemann, Great Britain. (2007)
The Manufacture, Storage and Import of Hazardous Chemical Rules, Government of India, 1989.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 102
Material And Safety Data Sheets
Section 1 – Product
Product Name Information RITUXIMAB
Emergency Overview
Rituximab is considered hazardous per the criteria under the OSHA Hazard Communication Standard (29
CFR 1910.1200). Adverse health effects have been observed in patients following intravenous (IV)
injection of therapeutic doses for treatment of non-Hodgkin's lymphoma, and for use in treating certain
types of rheumatoid arthritis. It derives its biotherapeutic benefit from a monoclonal antibody (rituximab),
a protein that is not well absorbed by inhalation or by contact with eyes, skin, or mucous membranes.
Although the health effects of occupational exposure to this product are not fully known or characterized,
no adverse effects are anticipated as a result of occupational or incidental exposure. This product is a clear,
colorless liquid.
Routes of Exposure
Direct contact with eyes, skin, or mucous membranes is the possible primary route of occupational
exposure. 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
~145,000 Daltons) and its poor potential for absorption
Formula (drug substance): Recombinant chimeric mouse/ human monoclonal antibody to CD20
antigen.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 103
Material And Safety Data Sheets
Flammability/ Explosivity
Not flammable or explosive. No special fire fighting measures.
Take proper precaution to minimize exposure by using appropriate personal protective equipment. If
material is released or spilled, soak up material with absorbent material and wash spill area thoroughly
with soap and water. Dispose of collected material in accordance with applicable waste disposal
regulations.
Section 7 – Handling and Storage
Refrigeration (2-8°C, 36-46°F) is advised to maintain longer pharmacological activity.
Protect from sunlight. Avoid agitation.
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, such as latex gloves and lab coat. Wash hands and other potentially exposed areas immediately
after handling material.
Eye Protection
As a prudent chemical hygiene practice, use safety glasses with side shields.
Other
Clean all protective equipment after use.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 104
Material And Safety Data Sheets
Stability: Stable
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 105
Material And Safety Data Sheets
This product is protein based and will rapidly degrade in the environment.
Dispose of waste residues according to prescribed federal, state, and local guidelines.
No additional information.
The above information is offered in good faith and with the belief that it is accurate.
While efforts are made to provide useful information relating to handling, in the event of
an adverse incident associated with this product, this Material Safety Data Sheet
(MSDS) is not, and is not intended to be, a substitute for consultation with appropriately
trained personnel.
Manufacture of 100000, 10ml fill vials per annum at 10mg/ml strength of RITUXIMAB Page 106
S001 B001 Seed Bioreactor1
P-1
FC001
Process Flow Diagram
P-34
Anil V. Vibhute