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Interferon
Hirak Desai, Douglas Cohen, Rushang Patel
Background
• Interferons (IFNs) are natural proteins produced by the cells of the immune system of most vertebrates in response to challenges by foreign agents such as viruses, bacteria, parasites and tumor cells.
• Extensively used in treatment of Hepatitis C and Multiple Sclerosis.
• Administered as a weekly injection.
Overall Operation ProcessCell Lysis
Extraction
Affinity Chromatography
Gel Filtration Ultrafiltration Refolding/ Anion Exchange Chromatography Gel Filtration
Final Product
Cell Lysis
• Uses Microfluidics HP-5000 Homogenizer.
• ln (1 – R) = -kNbPa • R = 0.98, N = 7• Flow rate = 950 ml/hr,
Pressure = 5000psi• 16.8g yield• 80.8L total volume
Protein Extraction• 7M Guanidine HCl
used to solubilize the protein.
• Guanidine/HCl used because it does not denature the protein, and results in an efficient extraction.
• Yields 16.5g of Protein.
Affinity Chromatography• Intracellular Interferon has a
Histidine –tag, which has affinity towards Nickel ions.
• By immobilizing nickel ions in a resin, the protein will bind to the resin packed in the column.
• Once isolated, the column is eluted with a step gradient of decreasing pH from 8.0 to 4.0 utilizing a 0.1 M glycine-HCl buffer containing 7 M urea.
• Yields 16.1g protein.
Gel Filtration/SEC• It separates solutes on the basis of their
size, and removes the small molecules within the system.
• Performed using Sephadex G-100 column
• First with 7M Urea and then with 7M Phosphate buffer.
• Proteins eluted in two peaks• Resulted in a much increased purification
factor.• Ultra Filtration step follows in order to
minimize time expenditure by reducing total volume
Anion Exchange Chromatography
• Before this step, protein is refolded with 0.1 M Trs-HCI pH 9 0, containing 10% glycerol
• Protein solution loaded onto a polybuffer exchanger.
• Reduces nucleic acids within the solution, which reduces potential contaminants.
• Active receptor monomer eluted with 0.35M NaCl solution.
Scale Up
• G1 (Q1/V1) d2p1 = G2 (Q2/V2) d2p2 • Scale Up Table
Step Q1 V1 Q2 V2
Nickel-ion Chelate Absorbent
50 mL/hr 100 mL 250 mL/hr 500 mL
Gel Filtration in 7M Urea
30 mL/hr 1000 mL 60 mL/hr 2000 mL
Anion Exchange Chromatography
6 mL/hr 200 mL 15 mL/hr 500 mL
Gel Filtration in Phosphate Buffer
6 mL/hr 200 mL 15 mL/hr 500 mL
Step Operation Volume [L] Interferon [g]
Activity [U/L]
Specific Activity
Percent Yield
Purification Factor
Processing Times [hours]
1 Cell Lysis 80.8 16.8 0.04 1 98 % 1 70
2 Guanidine/HCl Extraction
20.2 16.5 0.32 4 96 % 4 5
3 Nickel-ion Chelate Absorbent (Chromatography)
4.04 16.1 109 272 94 % 272 13.5
4 Gel Filtration in 7M Urea
2.02 11.0 126 316 64% 316 33.7
5 Ultra Filtration
0.25 10.8 15600 321 63% 321 7
6 Re-folding/ Anion Exchange Chromatography
0.20 5.14 15600 7778 30% 7778 13.3
7 Gel Filtration in Phosphate Buffer
0.18 4.80 16600 8333 28% 8333 12
Health and Environmental Issues
• The most frequent side-effects are flu-like symptoms: increased body temperature, feeling ill, fatigue, headache, muscle pain, convulsion, dizziness, hair thinning, and depression.
• These are usually reversible and disappear a few days after the therapy has ended.