bel 702 final report.docx
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Production of 170 gm ofIFN-
SUBMITTED BYDEVANSH DURGARAJU (2009BB50009)
MAY 10, 2013
BEL 702 Desi n Re ort
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1. Introduction.
1.1 Product Details
Interferons (IFNs) are produced by the body after a viral infection to limit the spread of
the infection, which is done by limiting the proliferation of the viral cells. IFNs are introduced
into the bloodstream to induce blood cells to produce an enzyme that limits the infection. Their
other useful functions include affecting cell differentiation, cell growth and antigen expression
on surfaces.
There are three types of inteferons. Namely, alpha, beta and gamma. IFN- is also called as the
leukocyte interferon. The main source of its production is from B-lymphocytes. IFN- is a
multifunctional immune-modulatory cytokine. It was approved as drug for treatment of hepatitis
C by the US FDA on February 25.
1.2 IFN- uses include:
1. Anti-viral application such as chronic Hepatitis C and Hepatitis B. These make up the
bulk of IFN- sales.
2. It has also been used for treatment of Hairy Cell Leukemia (HCL) and AIDS related
angiogenic tumors.
1.3 Companies involved in IFN- production:
Commercially, IFN- is marketed under the brand names Alferon, Roferon, Intron and Welferon
in the USA and Canada. They are produced by commercial processes to replace naturally
produced interferons in the body to fight infections. The following is a list of some companies
that produce IFN- in some form or the other:
Hoffmann-La Roche Inc.
Biogen Idec Inc.
GTC Biotherapeutics Inc.
NaPro Biotherapeutics
Promega
Anogen
Endogen
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2. Outline of Technology
Production of IFN- is done using B-Lymphocytes. The production of IFN- is
stimulated by activators like the endotoxin LPS, Dendritic Cells etc. [ Wang et al2012 ] In
general two major phases of the production of IFN- are present:
Isolation of DNA for IFN- from Lymphocytes.
Expression of this Gene in a foreign cells (CHO cells, Yeast,E.coli)
In this design project CHO cells were chosen as the expression cells. [ Chusainow et al2008 ]
The flowchart below shows the total process outline.
Chart 2.1 - Outline
Upon infection by virus, the interferons are released into the bloodstream. To further stimulate thesecretion, we use activators like endotoxin LPS. After 12-18 hours of stimulation, the lymphocytes are
lysed and RNA will be isolated. [ Wang et al2012]
After RNA isolation, the real time RT-PCR is done to convert RNA to cDNA, also specific primers are
used so that only the gene of IFN- is amplified.
Isolate IFN-
gene
Ligate into
Vector
Transfect into
CHO cells
Grow in Batch
Reactor
Elute IFN- in
Ni-Affinity
column
Centrifuge Ultrafiltration
Freeze-drying
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After which, the isolated gene is inserted into a vector and is transfected into the CHO cells. A
batch is then used to grow the cells and make them express the protein of interest.
The vector being used is pSecTag2/Hygro by Invitrogen. A figure of the Vector is shown
below. [ Invitrogen Website]
F igure 2.1 - Vector
2.1 Batch Reactor
Fermenter of 1200 L volume is used. The nutrient that is being used here is HYQ-PF-CHO. It isa protein-free mixture of nutrients and salts, thus decreasing level of contamination in the protein
of interest from external proteins. [ Kallel et al2002 ] Additionally Oxygen and Water need to be
supplied.
The temperature of the batch reactor is maintained at 300 C and the pH is between 6.8 and 7.2.
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2.4 Ultrafiltration.
In this type of membrane filtration, we use hydrostatic pressure to force a liquid against a
semipermeable membrane, which separates the particles on the basis of their sizes. Solids and
solutes of high molecular weight get retained and water and low molecular weight solids will
pass out through the membrane.
The UF membranes are anisotropic structures consisting of a thin, dense skin supported by a
micro-porous membrane sub-structure.
A figure of a membrane filter is shown below
F igure 2.2
This UF process follows the centrifugation process and it helps in the removal of water and the
low molecular impurities.
2.5Lyophilizer.Lyophilization or Freeze drying is a dehydration process used in making a material more
convenient for transport by reducing it to a powdery form rather than its native liquid form.
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The process involves freezing the whole material and then reducing the surrounding pressure to
allow the frozen water to sublimate directly from the solid phase to gas phase. Thus the IFN- is
reduced to a powdered form and thus can be transported and sold easily.
3. Process Kinetics and Mass Balance
Our energy source here is HYQ-PF-CHO, which contains all needed nutrients.
It is assumed that the reactor is charged with only one CHO cell initially. So the intial cell
concentration is 1/1000 L = 10-6 cell/ml. [83 % of total volume is usable, hence 1000 L from
1200 L]
It takes 1.2 days to reach the maximum cell concentration (2.8 x 106 cell/ml) [Assumed value]
Hence the amount of cells produced is 2.8 x 1000000 x 10
6
= 2.8 x 10
12
cells
We assume that the yield of protein per cell is 50 x 10-12 gm/cell.days [Assumed]
Hence, the Amount of protein produced per batch is = 168 gm.
Days per cycle = 2(Batch reaction) + 7(Downstream) + 1(Cleaning) = 10 days.
Ideally we can have 310-320 days per year on which the plant can function.
Hence we can have 31-32 Cycles per Year.
Which gives us a yield of around 31 x 168 = 5.2 Kg of IFN-alpha per year.
A considerable amount of wastage is assumed and thus this number is rounded off to 5 Kg/year.
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4. Flowsheet.
F igure 4.1 - F lowsheet
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Table 4.1F lowsheet Flows
Compound
Str. No.
( Kg/Batch)
1 2 3 4 5 7 8 9 10 11 12 13 6
Str. No
Compou
(Kg/Batc
Biomass
1E-
12 0 0 3.22 - 3.059 0.161 0.145 0.0161 0.0074 0.0087 0 0.0087 Biomas
Product 0 0 0 0.194 - 0.0097 0.184 0 0.184 0 0.184 0 0.184 Produc
Media 0 3.5 0 0.353 - 0.335 0.0176 0.0176 0 0 0 0 0 Media
Water 0 996.2 0 996.2 - 996 9.5 7 2.5 2.25 0.25 0.245 0.005 Water
Oxygen 0 0 0.27 0 - 0 0 0 0 0 0 0 0 Oxygen
Total
1E-
12 999.7 0.27 1000 - 999.403 9.86 7.162 2.700 2.257 0.4427 0.245 0.1977 Total
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5. P & I Diagram.
The sizes of pipes can be calculated because all the flows are known. The standard pipe sizes
were found from the Perrys Handbook tables. The schedule number and the nominal pipe size
are both given against the particular pipe in the P&I diagram. The diagram is given below.
F igure 5.1P & I Diagram
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6. Sizing of Pipes
Table 6.1Pipe Sizes
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7. Major Unit Operation design. Pump Design.
The centrifugal pump is generally preferred because of its low maintenance, simplicity and low
cost along with its quiet operation sound. A centrifugal pump is preferred for streams with low or
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no biomass. But for high biomass flows we prefer a peristaltic pump. This done to prevent
choking/clogging of the pump. The specific speeds of the important pumps in the P&I diagram
have been tabulated in the table below.
Table 7.1
Pump Specif ications
Pump GPM RPM L/H Ns
P01 0.026 1750 5.952 167
P02 0.000259 1750 0.059 16.7
P03 0.00007 1750 0.016 8.7
P04 0.0000132 1750 0.003 3.7
Calculation methodology used:
Ns = NQ0.5/H0.75
Where, Q = flow rate in gpm
H = Head Loss in meters
N = 1750 RPM
8. Mechanical Design
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Mechanical design has been done in the supplementary pages provided. The following table is a
summarization of the values of various vessel dimensions.
Table 8.1
Mechanical Design Summary
9. List of equipment used and specifications.
Shell
Volume of batch fermenter 1
M
Height 2.53 m
Internal diameter 0.79 m
Thickness of shell 0.019 m
Weight of vessel 865.15 kg
Closures
Crown radius 0.606 m
Knuckle Radius 0.04 m
Thickness of torispherical head 0.0076 m
Nozzle Flange and Gasket
design
Internal diameter of nozzle 0.017 m
Thickness of nozzle 0.0021 m
Outer diameter of nozzle 0.0213 m
Internal diameter of gasket 0.022 m
Outer diameter of gasket 0.026 m
Number of bolts 4
Diameter of bolt 0.0056 m
Thickness of flange 0.0078 m
Diameter of flange 0.0392 m
Skirt and Skirt Bearing
Plate
Height of skirt 0.8 m
Thickness of skirt 0.00009 m
Inner skirt diameter 0.82 m
Outer skirt diameter 0.828 m
Thickness of skirt bearing plate 0.0015 m
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1. Fermenter
After the Mechanical Design of the vessel the following specifications are available for
the fermenter:
Material of Construction = Stainless Steel
Volume = 1 m3
Height = 2.53 m
Diameter = 0.79 m
Thickness of Shell = 1.9 cm
2. Affinity Chromatograph
The affinity chromatograph, as described earlier, is used to separate the protein of interest
from other proteins. It utilizes the binding of the his-tagged protein to the Ni+ ions on
the Sepharose Fast Flow Resin.
Resin = Ni Sepharose 6 Fast Flow from GE Healthcare Life Sciences.
Binding: 20mM sodium phosphate, 0.5 M NaCl, 20-40 mM imidazole.
Elution: 20mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole.
3. Ultrafiltration Unit
The UF membrane is used to remove water and low molecular weight particles from the
product to get to a drier product. The generally used ones are hollow fiber UF membranes
for such operations. Commonly used material for preparation of membrane is
polysulphone. The membrane is Semipermeable. It also has a support material layer.
Every UF unit has a MWCO (molecular weight cut-off, it is around 10kDa), particles
below this MWCO can pass through the membrane.
10. Cost Analysis
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Chart 9.1Organizational h ierarchy
Table 9.1Salary of Employees
Employee Annual CTC (in Rs.)
Chairman 2000000
CEO 1500000
Plant Manager 1400000
Sales Manager 1400000
Production Manager 1000000
Quality Manager 1000000
Packaging Manager 800000
Asst Manager and Skilled Asst. (x11) 5500000
Total Expenditure 14600000
Equipment Purchase Cost Installation Cost Maintenance
cost(annual)
Fermenter 53537000 26768000 5353000
Chairman
CEO Plant Manager
Production
Manager
Asst. Manager
(x5)
Quality Manager
Skilled Asst.(x3)
Packaging
Manager
Skilled Asst. (x3)
Sales Manager
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Affinity
Chromatograph
19054000 952000 1905000
Centrifuge 4968000 2484000 496800
Ultrafiltration 6134000 2345000 543200
Lyophilizer 105192000 52596000 10519200
Total Expenditure 1135605000 85145000 18817200
Table 9.2Equipment Cost
Other than the main equipment that was covered in the above table, there are also subsidiary
equipment that are used in the process. The cost of these are assumed to be various fractions of
the Purchase Cost (PC). PC = 1135605000 Rs.
Table 9.3Other Costs
Other Costs Cost fraction x PC Cost
Piping 0.3 x PC 340681500
Instrumentation 0.35 x PC 397461750
Buildings 0.4 x PC 454242000
Electrical Facilities 0.1 x PC 113560500
Total Expenditure 1.15 x PC 130595750
The above two tables were the fixed cost that are related to the establishment of the plant (
Except for the Maintenance cost). Say, the plant takes 3 years to get to a functional stage to start
actual production.
The total expenditure incurred in these 3 years is calculated:
Equipment cost (Purchase + Installation) + Other Bulk Cost = 1135605000 + 85145000 +
130595750 = 2526695750
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The maintenance cost of the Plant are felt financially earlier than the start of the plant. This is
assumed to be after 2 years of setting up the plant.
Other miscellaneous expenses to do plant planning in the first 2 years is taken as = 5881720 Rs.
Now,
Yearly Cost = Annual Operation Cost + Annual Maintenance cost.
We get the following Cash flow Diagram for the process. The cost of IFN-alpha is taken around
75$ per microgram. This is a very very high and thus Breakeven is reached pretty quickly in the
5th year. It is assumed that 100 gm of IFN is sold from the 168 gm produced each year.
F igure 9.1Cash F low Diagram
11. Summary of Design.
-3E+09
-2.5E+09
-2E+09
-1.5E+09
-1E+09
-5E+08
0
500000000
0 1 2 3 4 5 6
Cash Flow Diagram
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The IFN-alpha production has been described and a theoretical design been presented above.
There are a fair number of purification steps that are needed due to the high degree of purity
required. We are able to produce 168 gm of IFN-alpha per year which is very high for a
biotherapeutic. They are also very expensive due to the cost of the equipment. The breakeven is
reached fairly quickly in this case. But this is faulty because equipment data for high purity
mammalian cell systems is not available easily and thus values have been assumed from those of
regular high volume process prices. Hence, we get breakeven quicker in this particular analysis.
This might not happen in the event of an actual application, wherein it could take 10-15 years to
breakeven.
12. References.
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Wang J.P, Zhang L, Madera R.F. . Plasmacytoid dendritic cell interferon-
production. BMC immunology 2012, 13:35
Kallel H. , Jouini A, Majoul S, Rourou S.. Evaluation of Serum Free media for growth
of BHK-21 cells. J Biotechnol, 2002 May 23; 95(3):195-204
Chusainow J, Sheng Y; Jessna H.M, Choo P. A Study of mAb producing CHO cell
lines. Biotech and Bioengg. Vol 102 Issue 4.. Oct 8 2008.
Hochuli, E.; Bannwarth, W.; Dbeli, H.; Gentz, R.; Stber, D. (1988). Genetic
Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal
Chelate Adsorbent.BioTechnology6 (11): 13211325
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