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24 Cards in this Set
- Front
- Back
CHAIN + FLOREY SCALE UP AND PURIFY PENICILLIN |
- Felming unable to stabilise and purify penicillin, this was achieved by Ernst Chain and Howard Florey at Oxford in 1939 - In 1941 penicillin was used for the first time on a a 43 year old man, started to recover, supplies ran out, died - 1942 the first person was saved by penicillin - Used any vessel they could fine to ferment penicillin - Fleming, Florey and Chain jointly awarded Nobel Prize in 1945 |
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OFF TO AMERICA |
- Wartime conditions made penicillin production difficult so Florey went to America - Here they developed deep tank culture methods using corn liquor as a growth medium - Production increased further when P.notatum was replaced by P.chrysogenum found on a rotting melon - In WW2 penicillin production was prioritised |
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COMMERICAL PRODUCTION OF ANTIBIOTICS |
- Antibiotics are secondary metabolites (not waste products) - Elaborate molecules produced in low amounts in nature - Overproduced in industry with approx 30,000 tons annually - Pfizer pioneered industrial-scale production in USA in 1949, production plants now located in the developing world for economics |
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GENERAL PRINCIPLES OF PENICILLIN PRODUCTION |
- Upstream processing: Preparation of growth medium and seeding culture of Penicillium (Fungal culture grown in a stirred-tank bioreactor known as a fermenter, penicillin is secreted into the medium) - Downstream processing: Extraction and purification of Penicillin |
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PENICILLIN PRODUCTION PROCESS: SIMPLE STEPS |
- Medium undergoes heat sterilization - Seed culture added and fermentation takes place - Biomass removal - Addition of solvent and centrifugal extraction - Extraction - Fluid bed drying |
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GROWTH MEDIA |
- Orginally, Flemming used simple media Czapek Dox agar - poor scale up - Florey + Chain tried: yeast extracts, casein digest, complex nitrogen sources, extracted oilseeds - Used of lactose instead of glucose increased yields due to slower growth - Breakthrough came when addition of 'corn-steep liquor' (CSL) increased yield 5-fold (plenty in USA). |
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CORN STEEP LIQUOR |
- For penicillium to grow medium must contain carbon source. This is provided by CSL and glucose - Source of phenylacetic acid - component of penicillin molecule - Also contains Magnesium sulphate, Potassium phosphate and sodium nitrate providing essential ions required for metabolic activity |
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STERILISATION OF FERMENTER AND GROWTH MEDIUM |
- Normally achieved by superheat steam at high pressure (121 degrees, 30 psi) - Sterilisation for short duration to minimised medium degration |
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BIOREATOR DESIGN |
- Can be enormous (250,000 litre fermentors used in industry) - Metal strips running up fermenter walsl: baffles - Air supply - Steam/cold water in and out passages to control temp - Passage to remove culture |
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PREPARATION OF PENICILLIUM SEED CULTURE |
4-7 days to prepare depending on size of the bioreacor |
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FERMENTATION CONDITIONS |
- Usually done in fed-batch mode, therefore glucose not added in high amounts at the beginning as can inhibit penicillin production - Penicillin secondary fungal metabolite, so fed-batch = high yields - 20-25 degrees, pH 6-6.5, pressure: 1.02atm (depending on funal strain/ bioreactor) - High pressure prevents contamination - Mixed by rotor 'impeller' - Stop run when at peak, usually 5-6 days in a 250,000L Bioreactor |
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CONTROL OF pH |
- Optimum pH = 6.5 - Maintained by carbonate/phosphate buffers - Monitor continuously, using autoclavable pH electrodes - Control pH by addition of sodium hydroxide solution |
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CONTROL OF TEMPERATURE |
- Penicillin yield very sensitive to temperature - Optimal at 25 degrees - Bioreactors 'jacketed' by hot/cold water |
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CONTROL OF FOAMING |
- Aeration and mixing causes frothing - Detected by foam sensors - Antifoaming agents added to control froth |
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CONTROL OF AERATION |
- Fungus P. cyrysogenum is aerobic - Needs large quanitities of oxygen - Achieved by sparging (air bubbled in) - Rate of supply of oxygen critical (depends on reactor size) |
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FILAMENTOUS CULTURE GROWTH |
- Interlocking strands of branching mycelium - Highly viscous pseudoplastic fluid - Difficult to stir and aerate - High shear may result in cell damage |
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PALLET GROWTH |
- Pellets 0.1mm to 1cm diameter - May occur if inoculum low and under some culture conditions - Easy to stire - Newtonian characteristics - Centre of pellet autolyses due to anoxia |
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SINGLE CELL GROWTH |
- Resembles yeast cultures 5-12 micrometers diameter - Developed as part of optimisation programme - True single cells - Easily stirred - Optimum gaseous exchange |
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REMOVAL OF BIOMASS |
- At end of fermentation biomass removed (pH rises to about 8.5) - Done by filtration usually with a rotary vacuum filter - Can run continuously and cope with high volumes - Non-oxidising acid such as phosphoric acid added to bring pH bacj to 6-6.5 to prevent loss of acitivty of penicillin |
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ADDING SOLVENT AND FURTHER FILTRATION |
- To dissolve penicillin in filtrate organic solvents such as amyl acetate of butyl acetate are added - Dissolve penicillin better than water at physiological pH - Penicillin now present only in solution and other solids considered waste - Waste solid separated from solution using DISC CENTRIFUGATION |
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EXTRACTION - PURIFICATION OF PENICILLIN |
- Acetate solution mixed with phosphate buffer - Chloroform extraction - Finally extrated with ether - Penicillin now present in high concentrations in ether and solution is mixed with sodium bicarbonate solution to obtain penicillin-sodium salt, this is stable powder at room temp - Penicillin-sodium salt obtained from liquid by basket centrifugation |
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FLUID BED DRYING |
- Necessary to remove any remaining moisture in the powdered penicillin salt - Hot gases pumped into chamber containing powdered salt inside a vacuum chamber - Dry powder now ready for storage and packaging - Activity of product determined in QC lab |
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YIELD OF PENICILLIN |
- Cultures give 30g/litre - Yield increasing 10,000 fold since Fleming |
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TOTAL SYNTHESIS OF PENICILLIN (NOT EASY) |
- Penicillin has an easily altered beta-lactam ring and three steroisomers with only one active - Not easy to make and obtained by total synthesis in small quanitites - Thus a combination of fermentation and organic synthesis must be used - But acylating purified penicillin G and substituting the 6-amide group, 6-aminopenicillanic acid can be made: many derivatives can be synthesised |