System For The Production Of A Protein Therapeutic Biology Essay

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Rheumatoid arthritis (RA) is a chronic auto-immune inflammatory disorder that causes pain, inflammation of the joints and swelling. It makes the joints feel stiff and the patient generally feels unwell and tired. The human immune system which is designed to fight various infections, attacks the lining of the joints instead, causing them to become inflamed. Eventually, RA can also diffuse into the pericardium, sclera, pleura and lungs, commonly in the subcutaneous tissue under the skin.

The local swelling or tissue lump in the skin, which is often subcutaneous, is a characteristic feature of RA. In lungs, fibrosis (presence of excessive collagen) is the chief response to the rheumatoid disease. Renal amyloidosis (abnormal deposition of amyloid proteins) can occur as a consequence of inflammation in the kidneys. People with RA are more prone to heart diseases like the atherosclerosis, with risks of myocardial infarction (heart attack).

There is no cure for Rheumatoid arthritis, as for most of the autoimmune diseases. But there are treatments available that can alleviate the symptoms or modify the disease process. Pain killers may relieve the current symptoms but have no long term effects, for example, they cannot stop the damage caused to joints. Disease modifying anti-rheumatic drugs (DMARD) reduces the rate of damage to bone and cartilage, for example methotrexate. But the use of DMARD's has many side effects. Some of them are:

Increase in risk of getting an infection, due to the drop in white blood cells.

Aching muscles, headaches, cough, sore throat, pain passing urine and feeling of cold are common side effects.

Breathlessness and tiredness are also common in some medications, and may require additional blood transfusions as there is a drop in red blood cells as well.

Getting bruises easily is another side effect due to drop in platelets.

Fatigue, taste changes, mouth sores and ulcers, diarrhoea, hair loss and thinning are other common side effects.

It is thus necessary to increase awareness of the symptoms so that the disease can be diagnosed at an early stage, which is not only beneficial but also the treatment has less side effects comparatively. An alternative therapy lies in the use of immuno suppressant protein therapeutics like cyclosporine.

Cyclosporine: an alternate protein therapeutic

Cyclosporine or Cyclosporin A is an immunosuppressant drug used to reduce the activity of the patient's immune system, to prevent the immune attack on joints while suffering from RA. It is an undecapeptide (composed of a chain of 11 amino acid residues) with anti-inflammatory effects. It also finds use as an immuno-suppressant, anti-fungal, and anti-parasitic drug.

Fig.2: Structure of cyclosporin A or cyclosporine. 11 amino acid residues can clearly be seen.

(Field S.Q., 2006)

Extensive screening experiments on fungi reveal that a variety of fungus species have cyclosporin producing properties. A few examples are Tolypocladium geodes, Trichoderma viride, Neocosmospora vasinfecta, Beauveria nivea and many more. The fungi Beauveria nivea (previously known as Trichoderma polysporum, Tolypocladium inflatum, and Tolypocladium niveum), which is a filamentous fungi is known to have the highest productivity of cyclosporin and thus can be used for large scale industrial production. About 500mg of cyclosporin A can be produced per litre of ferment. Cyclosporin can be produced by submerged culture fermentation, static fermentation, solid state fermentation and also enzymatically. Solid state fermentation is best used platform for the production of cyclosporin.

Solid state fermentation (Apparatus and working)

Solid state fermentation is a fermentation process in which micro-organisms are grown on solid materials without the presence of a free liquid. The moisture necessary for microbial growth exists in an absorbed state or complied within the solid matrix (Krishna C., 2005). The moisture level is maintained at or above 12% as below this level, biological activities cease to exist. The solid state fermentation process is of two types: natural and pure culture solid state fermentation, out of which pure cultures are industrially preferred as they help in optimum substrate utilisation for the targeted product (cyclosporin in this case).

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Fig.3: Solid state fermenter.

Closing lid.

Air outlet.

Orifice for inoculation of the fermenter.

Module bases permeable to air and water.

Taking up of substrate for cultivation of organism.

Cooling devices.

Air inlet.

Water inlet.

Orifice for letting out water.

Source: Patents online

The oval tank would have a capacity of 50 litres that can be closed on top by a lid. The vessel with a diameter of 30cm and a height of 70cm is impermeable to air and water and contains module bases enabling them to take up cultivation substrate. The outer shell of the fermenter is made of heat resistant glass. Eight module bases were mounted with 8cm gap between them. The cooling devices allow the evacuation of heat of the reaction from the substrate. The bottom contains an inlet for sterile, moistened air which circulates throughout and leaves through the air outlet mounted on the lid. The fermenter is initially filled with sterile water in order to provide sterile cultivating conditions for the microorganism. The suspension of microorganisms or inoculum, which consists of highly cultured suspension of small germinable units like spores or conidia's of the micro-organism, is then introduced.

Materials required

In the first step, various agricultural wastes and oil cakes were screened for maximum production of cyclosporin A. Also the effect of hydrolysis of starchy substrates, combination of different solid substrates and initial moisture content need to be determined. The effects of additional supplements such as salts, C-sources and N-sources should also be investigated in order to achieve maximum production. Glucose, sucrose, maltose, glycerol, yeast extract, agar, malt extract, mycological peptone, casein peptone, ammonium sulphate, sodium nitrate and urea to provide essential nutrients like carbohydrates and proteins necessary for growth.

Salts like magnesium sulphate, ferric chloride, sodium chloride and solvents like NaCl, HCl, acetonitrile, n-butyl acetate, NaOH, etc. can be used as supplements for the medium. Well known fermentation medium for growth of the strain is Wheat bran and rice bran.

Conidial suspensions of about 108 per ml from the strain Beuveria nivea can be prepared and used as an inoculum. The culture conditions in the fermenter were maintained at 26 degree temperature and70% initial moisture to promote growth. Cyclosporin A is produced as a secondary metabolite during the stationery phase, as a response to stress conditions.

Optimisation of fermentation conditions

Time: Takes about 9 days to give maximum yield, decreases gradually.

Substrate: Wheat bran can be used to obtain maximum yields (1031mg/kg of wheat bran (Murthy R et al., 1999).

Extracting solvent: acetone, methanol, butyl acetate and toluene.

Moisture content: 70% moisture content is known to show maximum production.

Effect of inoculum age: Inoculum incubated for 10 days shows maximum results.

Effect of incubation temperature: 25 degree Celsius.

Effect of initial pH of the supplement: pH 2.

Cyclosporin A extraction and estimation

The ferment extract can be extracted by 40ml of butyl acetate at 25oC and centrifuged at 180 rpm for 24 hours. The extract will then be filtered using Whatman filter paper No.1 and a Pall 0.2µm membrane filter like Nylon 6 and Ultipor N55 to obtain a brown coloured extract. The extract was dried and dissolved in HPLC grade acetonitrile. 20µl of the sample can be analysed using HPLC to test the concentration of Cyclosporin. Acetonitrile and water in the ratio of 70:30 can be taken as mobile phase, temperature of the column maintained at 70oC and the HPLC profile monitored at 210nm.

Advantages of using solid state fermentation over submerged culture fermentation

Solid state fermentation has the following advantages:

Use of agro-industrial residues as substrate that supplies all the nutrients required for growth.

It gives higher yields as compared to the traditional submerged culture technology.

Provides a natural habitat for fungal growth.

It is relatively environmental friendly as it lets out fewer pollutants.

It requires a lower investment capital.

Advantages of using Cyclosporine as a protein therapeutic

A high grade immunosuppressant that treats inflammation effectively and does not allow the immune system to attack joints causing RA and yet leaves immune system with enough immune activity to fight other infections.

Easy to obtain medium, suitable and economical to produce high yields from an industrial point of view.

Lesser side effects than other disease modifying anti-rheumatic drugs (DMARDs).

A potential multi-purpose drug with anti-fungal, anti-parasitic, anti-inflammatory and immuno-suppressive properties. Over USD 1 billion market for the use as immuno suppressive drugs.

Potential limitations to the feasibility of the project

First and the foremost limitation would be to gather appropriate funds to setup the apparatus and office. For this I would need to prepare a viable business model stating my business strategies so as to obtain collaborations with companies for my project.

Limitations of using solid state fermenters:

Solid state fermenters lack free water, thus smaller fermenters have to be used.

Difficulties in agitation of the substrate bed which results in unevenly distributed chemical environment in the substrate bed.

Difficulties in fermentation control especially due to heat build up and control of aeration and moisture.

Difficulties in rapid determination of microbial growth.

Limitations of using cyclosporin A as a protein therapeutic:

Although shows less side effects than the other drugs currently in market, but cannot neglect the fact that it has certain side effects like increased hair growth and trembling and shaking of hands. Long term therapy could cause an increased blood pressure and kidney problems.

Does not cure rheumatoid arthritis completely.

Conclusion

There is a growing need in the market for drugs that can cure RA completely. If not curing the disease completely, drugs currently used in the market only alleviate symptoms of the disease and often result in the acquiring of another infection, due to side effects caused by drugs. Cyclosporin A is a DMARD that comparatively causes fewer side effects than the other DMARDs used. Hence, safe large scale production of cyclosporin A might be of demand.

The filamentous fungi Beauveria nivea produces cyclosporin A in maximum concentration and hence been utilised in my project. Moreover, the use of solid state fermentation to grow the fungi has shown to be advantageous than any other fermentation method. The solid state fermentation is not very popular in the West which should give me an edge over other companies producing the same drug.

Keeping the above factors in mind, large scale production of cyclosporin A as a protein therapeutic by solid state fermentation will lead to a commercially viable product.

Approximate word count (excluding figures): 1962.

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