Large-Scale Penicillin Production

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CHAIN + FLOREY SCALE UP AND PURIFY PENICILLIN

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- 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

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- 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

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- 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

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- 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

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- 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

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- 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

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- 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

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- 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

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- 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

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4-7 days to prepare depending on size of the bioreacor

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FERMENTATION CONDITIONS

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- 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

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- 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

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- Penicillin yield very sensitive to temperature

- Optimal at 25 degrees

- Bioreactors 'jacketed' by hot/cold water

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CONTROL OF FOAMING

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- Aeration and mixing causes frothing

- Detected by foam sensors

- Antifoaming agents added to control froth

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CONTROL OF AERATION

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- 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

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- 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

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- 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

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- 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

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- 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

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- 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

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- Acetate solution mixed with phosphate buffer

- Chloroform extraction
and mixed again in phosphate buffer

- 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

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- 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

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- Cultures give 30g/litre

- Yield increasing 10,000 fold since Fleming

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TOTAL SYNTHESIS OF PENICILLIN (NOT EASY)

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- 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