bel 702 final report.docx

7/29/2019 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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