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.