Antibiotic Production Although Fleming discovers penicillin in 1929, large scale processes for its production were not

realised until the early 1940s. The treatment of wartime casualties became a priority and the work of Florey realised the potential of this drug in the battlefield. Initially penicillin was produced in milk bottles because the technology existed for the handling and filling of these vessels. Over time it became apparent that deep tank fermenters be used as they resulted in a great leap forward in process efficiency and productivity. Valuable lesson: Scientific discovery is not the singular catalyst to bring about significant change. It is also essential that suitable technology be proposed to exploit the potential to its full commercial impact. The early success in isolating a medically useful compound from a microorganism lead to the massive ongoing hunt for antibiotics that continues to the present day. Today, over 5000 compounds have been isolated, by far the largest proportion have been isolated from streptomyces cultures.

Of this only a relatively small number of compounds have been commercially successful in therapy, with hundreds of tonnes of compound being produced each year by fermentation. Penicillins and cephalosporins account for approximately 70% of market share. These are used either in their natural form or as semi synthetic derivatives. Antibiotics are produced generally as a result of some environmental stress that triggers a metabolic response. Antibiotics are produced as a defense mechanism to prevent the proliferation of other organisms when environmental conditions are challenging. The organism will incur significant metabolic overhead systhesising these compounds, so only does so under environmental stress. This is an important consideration when considering process design.

Production Processes Almost all antibiotics are produced in mechanically agitated and aerated vessels, the design of which is of crucial importance to the process. Media used for production are extremely varied and most contain both complex nitrogen and carbohydrate sources. Each production process is generally divided into the following key stages.

Culture Preservation Innoculum Preparation Seed Stage Production Stage Harvest, Extraction and Purification

Culture Preservation The preserved culture is a valuable asset and as little should be used as possible to initiate the process. Generally this preserved stock is in the form of inert spores. Spores can be stored in dry soil, lyophilised or frozen in a preservation solution.

Production Strain A high yielding strain is a prerequisite of any good antibiotic producing process. Continuous modification of process conditions will result in steady increases in yield however, large steps forward generally result from the introduction of a superior strain. Such strains are generally obtained through genetic manipulation. Random mutagenesis and subsequent screening are key strategies in strain yield improvement. Modern techniques of genetic engineering has seen the isolation and cloning of gene cartridges associated with the metabolic pathway for biosynthesis of antibiotics into non producing organisms with more desirable growth attributes. Remember if this approach is to be successful, then the means of inferring resistance to the antibiotic also has to be provided to the host microorganism. Scale Up Scale up is an important part of many cell driven processes and no more so than in antibiotic fermentations. Starting off with a small volume of a preserved culture, it is not feasible to inoculate your production fermenter immediately with this material. It is essential to amplify the starting innoculum

through a series of ever increasing volumes until enough material exists to innoculate the production system. In bacterial and yeast systems where vigorous growth is anticipated, 1:20 to 1:100 pitches are common. A common chain would be 1ml 100ml 10L 1m3 - <1000m3 Innoculum resuspension shake flask pilot plant fermenter seed fermenter production fermenter. For antibiotics the production vessel is generally in the range of 10-300m3 depending on the value of the final product. While the overall aim of the process is to have the microorganism producing antibiotic for as long a time as possible, it is always essential to consider that the catalyst has to be grown prior to its use. Low biomass concentrations in the production reactor may have deleterious impact on process timescale. Also consider our earlier statement, that antibiotic production is not growth associated and is as a result classified as a secondary metabolite. Biocatalyst growth and and antibiotic production are mutually exclusive. The solution to this issue is to have a growth medium which is used throughout the scale up process and have a production medium which is used in the final

stage of the process. The growth medium is formulated to encourage biomass growth to the detriment of antibiotic production and the production medium (in conjunction with process conditions) simulates the required environmental stress required for the synthesis of antibiotics. Production process Process economics key, one of the major process costs associated with the sterilisation of large volumes of fermentation medium. Continuous sterilisers often used due to superior efficiencies. The production reactor must be designed with the following considerations in mind (these considerations also apply to a lot of other processes):

High gas liquid mass transfer coeffients must be obtained. High interphasial nutrient transfer to ensure that nutrients are rapidly distributed and supplied to the organism. (organism growth morphology is a significant consideration here) Good bulk liquid mixing characteristics to ensure homogeneity within the vessel. Fermenters produce heat (through dispersion of kinetic energy and metabolism), therefore the vessel must be a good heat exchanger.

The vessel must be able to run aseptically for prolonged periods of time. (maintenance of positive pressure within vessel).

Most production fermenters in antibiotics are of traditional design. (For AS4s)

Stainless steel construction Vessel Height to Tank Diameter 2-4:1 One Rushton Impeller per Tank Diameter in Height. Air sparged into the vessel below bottom impeller between 0.3 and 1.5 VVM. Power consumption in reactor in the order of 1-4 W/L

Production Media A whole family of production media are required for an antibiotic producing process. Each medium is designed to support a particular stage of the process Media designed for the early stage of the process will be generally focussed on achieving optimal spore germination and strong vegetative growth. As antibiotics are secondary metabolites, the final production medium will be focussed on the over production of the antibiotic often to the detriment of growth.

Most media used for production are complex in nature. Although many organisms will produce their indigenous compound on chemically defined media, complex media are cheaper to formulate and have higher yields and productivities. As with any general growth media it must supply all the basic constituents for growth/product formation.

Carbon source Nitrogen Phosphates Trace Elements

In the case of antibiotic fermentations, the use of specific precursor molecules in media formulations is significant. For example: The production of Penicillin G is greatly improved through the introduction of Phenyl-acetic acid as a precursor into the fermentation medium. Nitrogen source, generally cheap supplements containing corn steep liquor (source of penicillin precursors: phenylalanine and phenethylamine), soya flour and fish meal. These materials may often supply unknown trace elements which aid production. Technical grade glucose and or starch can be used as a carbohydrate source. If the process is batch, one generally finds a mix of carbohydrates present.

Simple carbohydrates to get the process moving and complex ones to simulate substrate (catabolite) limitation. Catabolite Repression Many antibiotics are not produced in the presence of excess carbon sources, especially glucose. In order to overcome catabolite repression, the addition of the carbon source to the culture must be carefully controlled. Fed batch is the most common approach. Lactose used historically as carbon source in batch cultures due to its slow rate of hydrolysis (no residual glucose in fermentation media post hydrolysis) The presence of excess nitrogen compounds or phosphates in the fermentation decreases antibiotic production severely. Feedback Regulation If penicillin is added to a culture of penicillin producing fungi, synthesis of the antibiotic is limited. Precursors Secondary metabolites are synthesised from primary metabolites. The efficient production of antibiotics requires a steady flow of their precursors. For example -aminoadipic acid is an intermediate in the pathway of lysine biosynthesis.

High levels of lysine in the media shuts off that pathway by inhibiting the first enzyme in that pathway. This results in a shortage of all the intermediates in the pathway including the key intermediate for penicillin. Therefore the presence of lysine strongly inhibits penicillin production. Production Process Outline- Penicillin Spores of P. chrysogenum are used to inoculate 100ml of growth medium in a 500ml shake flask. 4 days incubation, contents are transferred to growth medium in 500L reactor. After incubation for three days, this culture is used to inoculate 180m3 reactor. Final Fermentation completed in 5-6 days. pH about 6.5 and the temperature is in the range 23-28C. Best current strains result in about 10% of the carbon in the glucose finds its way into penicillin G whose final concentration may reach almost 30g/L

Penicillin Variants Most fungi that produce penicillin can easily incorporate a variety of compounds into the acyl portion of the penicillin molecule. Over 100 variants have been produced and each has been evaluated for a variety of desirable attributes. All compounds must have the initial beneficial traits of Penicillin G along with additional advantages.

Acid Stability (allowing for oral ingestion) Lowered allergenicity 8% of population allergic to Penicillin G. Greater resistance to penicillinase

Penicillin V Increased acid stability. Allowing for oral ingestion. Penicillin O lower allergic sensitivity. Semi-Synthetic Variants The compound 6-aminopenicillanic acid can be readily acylated by chemical means to produce semisynthetic variants. Tens of thousands of compounds have been synthesised, resulting in new antibiotics with desirable attributes.

Down Stream Processing of Penicillin Broth contains 20-35g/L of penicillin Mycelia are separated from the liquid by filtration occasionally using filteraids or precoats Penicillin rich filtrate is cooled to 4C to minimise chemical and enzyme degradation during solvent extraction Sometimes the filtrate is further clarified by a second filtration with 1-1.5% Hyflo (a filter aid) sometimes with the precipitation of proteinaceous material by addition of aluminum sulphate or tannic acid. In the next stage of the process, penicillin is extracted into amyl acetate or butyl acetate using a continuous countercurrent process. Penicillins are strong acids with pKa values in the ranve of 2.5-3.1. As the acid forms are soluble in many organic solvents, they are extracted with high efficiency into amyl acetate or butly acetate at pH 2.53.0 The extraction is performed in continuous countercurrent multistage centrifugal extractors. Efficient extraction uses a solvent to broth ratio of 0.1 One such device is a the Podbielnak extractor. It consists of a cylindrical drum contining perforate

concentric shells and is rapidly rotated on the horizontal shaft (2000-5000rpm) Liquids enter through the shaft; heavy liquids is led to the centre of the drum and lighter liquid fed to the periphery of the drum. The heavy liquid flows radially outwards displacing the light liquid inwardly and both are led out through the shaft. These extractors are ideal for liquids with small density differences where short residence times are essential. The penicillin containing solvent is then treated with 0.25-0.5% carbon to remove pigments and other impurities. Penicillin is then back extracted into water by the addition of alkali (potassium or sodium hydroxide) or buffer at pH 5.0-7.5 The volume ratio of water to solvent in this stage of the process is 0.1-0.2 in continuous multistage extractor. Penicillin can be precipitated form the resultant aqueous phase. Penicillin crystals are washed and predired with anhydrous l-propanol, n-butanol or other volatile

solvent. Final Drying is accomplished using vacuum, warm air or radiant heat on large horizontal belt filters. Other Antibiotics Produced by Fungi Cephalosporins Molecule is relatively similar in structure to penicillin. Produced from selected strains of Cephalosporium Chemical structure is less susceptible to hydrolysis of the beta lactam ring than penicillin, by Staphylococci, but are readily hydrolysed by enzymes from gram negative bacteria. Fermentation processes similar to those employed for penicillin. Hoeever, even after extensive investigation, yields are considerably lower that those obtained in penicillin fermentation. Several semi-synthetic derivatives of Cephalosporin in commercial production.

Streptomycetes Actinomycetes represented by Streptomycetes, include more that thirty genera of gram positive bacteria that show branching, filamentous or irregularly rod shaped morphology. The term acintomycete first originated in 1887, coming from the Greek root, Ray Fungus However, their taxonomic position is now well established within the the kingdom of the Prokaryote. That said, Streptomycetes are boundary organisms from a morphological, physiological and metabolic point of view. The outstanding property of Streptomycetes is their ability to synthesize a variety of antibiotics. To date over 6000 antibiotics of microbial origin have been isolated. 60% of actinomycete origin, the rest from fungal and bacterial sources. Out of these 70 compounds have found practical application in human and animal medicine. 90% of these compounds come from the Actinomycetes. Antibiotics from Streptomycetes include almost all known structural classess of commercially important antibiotics.

Impact of Antibiotics Produced by Actinomycetes on Human Health Tuberculosis, caused by Mycobacterium tuberculosis, serious disease until the advent of streptomycin. Enterotyphus, 50% mortality rate, cured by chloramphenicol, Syphilis and bacterial dysentery are now only encountered rarely. 1946-1980 Average life expectancy of the Japanese male increased from 43 to 73. Much of this increase due to antibiotics. Infant mortality down to 1% of what it was in 1950. In addition to the obvious medical benefits, antibiotics help stabilise food supply by controlling animal, fish and plant diseases, leading to higher agricultural productivity.

Genetic Approaches to Antibiotic Process Optimisation In the last several years, important advances have been made in the technical procedures for genetic recombination and gene cloning in Streptomyces. However, most antibiotic synthesis processes are complex multistep processes, often poorly understood. Therefore, random chemically induced mutations continues to be the most widely applied and successful genetic procedure to improve the antibiotic productivity. In spite of the enormous economic importance of chemical mutation on antibiotic productivity, little is known about the fundamental mechanism of mutation. Mutations and Genetic Instability Spontaneous mutations include a variety of molecular insults, including deletions, duplications, transpositions, insertions, base pair substitutions and reading frame shifts.

Induced Mutations

UV raditation Chemical Mutations 4-Nitroquinoline-1-oxide Hydroxylamine Methyl Methanesulfonate Ethyl Methanesulfonate N-methyl-N'-nitro-N-nytrosoguanidine

Mutagenesis and Strain Development Mutation induction in Streptomyces is a complex process. Not all mutagens are capabile of inducing a high level of mutation in all streptomycetes.

Treat spore suspension with chemical or UV mutagen. Isolate improved mutants using screening techniques.

Shake Flask Screens are the most popular method for isolation of improved mutants and is still used frequently today.

Treat with mutagen Plate out on growth medium at low density Use agar plugs to isolate single colonies Innoculate liquid cultures with test colony. Screen for increased productivity.

Plate Based Techniques

As before, treat spore suspension and plate at low density. Culture the spores into colonies on the plate. Overlay the colonies with membrane innoculated with test organism. Zones of exclusion are measured using image analysis. Largest zone of exclusion, highest concentration of antibiotic diffused into the surrounding agar.