BEL 702 Design Report

Production of 170 gm of IFN-

SUBMITTED BY DEVANSH DURGARAJU (2009BB50009)
MAY 10, 2013

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

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 al 2012 ] 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 al 2008 ] The flowchart below shows the total process outline. Chart 2.1 - Outline

Isolate IFN- gene

Centrifuge

Ultrafiltration

Ligate into Vector

Elute IFN- in Ni-Affinity column

Freeze-drying

Transfect into CHO cells

Grow in Batch Reactor

Upon infection by virus, the interferons are released into the bloodstream. To further stimulate the secretion, we use activators like endotoxin LPS. After 12-18 hours of stimulation, the lymphocytes are lysed and RNA will be isolated. [ Wang et al 2012] 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.

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]

Figure 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 is a protein-free mixture of nutrients and salts, thus decreasing level of contamination in the protein of interest from external proteins. [ Kallel et al 2002 ] 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.

The fermentation reaction goes on for 1.2 days to grow and then produce IFN-. The IFN- is secreted out by a secretion system of the cell and hence lysis is not required. 2.2 Affinity Column

A His Tag is added to this gene to facilitate the separation of the resultant fusion protein from the reaction mixture. The separation is done in a Nickel Affinity column. The separation is based on the specific interactions between the his tags and the ligands. His-tagged proteins have a highly selective affinity for Ni+2 ions. Thus the protein of interest, which in our case is tagged with His will be selectively held back by the column and can be eluted out afterwards by changing the buffer conditions. [ Hochili et al 1988 ] Buffers used: [ GE Healthcare ] Binding: 20mM sodium phosphate, 0.5 M NaCl, 20-40 mM imidazole Elution: 20mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole

Resin used: [ GE Healthcare ] Ni Sepharose 6 Fast Flow from GE Healthcare Life Sciences.

Resin is regenerated using 50 mM EDTA in 20 mM sodium phosphate with 0.5 M NaCl and the ph =7.4. Upon final elution with the recommended elution buffer, the elution will contain his-tagged protein which is bound to imidazole, the his-tag has no effect on protein function, so need not be removed. But, the imidazole should be removed to minimize the effect it has on functionality of protein. [ GE Healthcare website]

2.3 Centrifuge. The final elute from the Ni Affinity column of his-tagged IFN- needs to be centrifuged to separate any remnant impurities in the elution. This step is commencing of the final purification of the IFN- .

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 Figure 2.2

This UF process follows the centrifugation process and it helps in the removal of water and the low molecular impurities.

2.5 Lyophilizer. 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.

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 106 = 2.8 x 1012 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.

4. Flowsheet.
Figure 4.1 - Flowsheet

Compound Str. No. ( Kg/Batch) 1 2 3 4 5 7 8 9 10 11 12 13 6

Str. No. Compound (Kg/Batch)

1EBiomass 12 0 0 3.22 3.059 0.161 0.145 0.0161 0.0074 0.0087 0 0.0087 Biomass

Product

0.194

0.0097 0.184

0.184

0.184

0.184

Product

Media

3.5

0.353

0.335 0.0176 0.0176

Media

Water

996.2

996.2

996

9.5

2.5

2.25

0.25 0.245 0.005

Water

Oxygen

0.27

Oxygen

1ETotal 12 999.7 0.27 1000 999.403 9.86 7.162 2.700 2.257 0.4427 0.245 0.1977 Total

Table 4.1 Flowsheet Flows

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. Figure 5.1 P & I Diagram

6. Sizing of Pipes
Table 6.1 Pipe Sizes

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

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 Specifications
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

Mechanical design has been done in the supplementary pages provided. The following table is a summarization of the values of various vessel dimensions.

Volume of batch fermenter Height Shell Internal diameter Thickness of shell Weight of vessel Crown radius Closures Knuckle Radius Thickness of torispherical head Internal diameter of nozzle Thickness of nozzle Outer diameter of nozzle Internal diameter of gasket Nozzle Flange and Gasket design Outer diameter of gasket Number of bolts Diameter of bolt Thickness of flange Diameter of flange Height of skirt Thickness of skirt Skirt and Skirt Bearing Plate Inner skirt diameter Outer skirt diameter Thickness of skirt bearing plate

1 2.53 0.79 0.019 865.15 0.606 0.04 0.0076 0.017 0.0021 0.0213 0.022 0.026 4 0.0056 0.0078 0.0392 0.8 0.00009 0.82 0.828 0.0015

M m m m

kg m m m m m m m m

m m m m m m m m

Table 8.1 Mechanical Design Summary

9. List of equipment used and specifications.

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

Chart 9.1 Organizational hierarchy

Chairman

CEO

Plant Manager

Sales Manager

Production Manager

Quality Manager

Packaging Manager

Asst. Manager (x5)

Skilled Asst.(x3)

Skilled Asst. (x3)

Table 9.1 Salary of Employees Employee Chairman CEO Plant Manager Sales Manager Production Manager Quality Manager Packaging Manager Asst Manager and Skilled Asst. (x11) Total Expenditure Equipment Purchase Cost Annual CTC (in Rs.) 2000000 1500000 1400000 1400000 1000000 1000000 800000 5500000 14600000 Installation Cost Maintenance cost(annual) Fermenter 53537000 26768000 5353000

Affinity Chromatograph Centrifuge Ultrafiltration Lyophilizer Total Expenditure

19054000

952000

1905000

4968000 6134000 105192000 1135605000

2484000 2345000 52596000 85145000

496800 543200 10519200 18817200

Table 9.2 Equipment 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 9.3 Other Costs Other Costs Piping Instrumentation Buildings Electrical Facilities Total Expenditure Cost fraction x PC 0.3 x PC 0.35 x PC 0.4 x PC 0.1 x PC 1.15 x PC 340681500 397461750 454242000 113560500 130595750 Cost

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

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. Figure 9.1 Cash Flow Diagram

Cash Flow Diagram

500000000 0 0 -5E+08 -1E+09 -1.5E+09 -2E+09 -2.5E+09 -3E+09 1 2 3 4 5 6

11. Summary of Design.

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.

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. BioTechnology 6 (11): 13211325

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