Penicillin extraction

The treatment of the crude penicillin extract varies according to the objective but involves formation of an appropriate salt, probably followed by treatment to remove pyrogens, and by sterilization. This last is usually achieved by filtration but pure metal salts of benzylpenicillin can be safely sterilized by dry heat if desired. 

Erythromycin is, like penicillin, isolated by solvent-extraction methods. It is an organic base, and extractable with amyl acetate or other organic solvents under basic conditions rather than the acidic ones that favor penicillin extraction. 

Derivation The mold is grown in a nutrient solution such as com steep liquor, lactose, or dextrose. After several days of cultivation the mold excretes penicillin into its hquid culture medium. This hquid is then filtered off, and the penicillin extracted and purified by countercurrent extraction with amyl acetate, adsorption on carbon, or other methods. Different varieties of penicillin are produced biosynthetically by adding the proper precursors to the nutrient solution. 

A number of industrial centrifugal extractors have been available since 1944 when the Podbielniak (POD) extractor, with its short residence time, was successfully applied to penicillin extraction (Barson and Beyer, 1953). In the POD, several concentric sieve trays are arranged around a horizontal axis through which the two liquids flow countercurrently. Liquid inlet pressures of 4 to 7 atm are required to overcome pressure drop and centrifugal force. As many as five theoretical stages can be achieved in one unit. 

This is just in contrast to another approach of modifying the molecular structure of peptidic compounds on the biosynthetic level, namely the directed biosynthesis of the desired compound structures by feeding of appropriate biosynthetic precursors or stimulating agents during fermentation, as it is routinely performed for production of penicillin V und G by the addition of phenoxyacetic acid or phenyl-acetic acid, respectively, as precursors to facilitate penicillin extraction from the culture broth. 

Common penicillin extraction solvents are amyl- and butyl-acetate or methy-lisobutylketone. As penicillin is extracted as a free acid at pFl 2-2.5 and the molecule is instable at this pFl, extraction occurs in a counter-flow using Podbielniak- and Luwesta-centrifugal extractors to shorten the contact time with the solvent and to prevent product decomposition. 

Immunogenic biological impurities mycelial proteins in penicillins extracted from broth Escherichia coli penicillin amidase in 6-aminopenicillanic acid used for synthesis of semisynthetic penicillins... 

Where settling is slow due to small density difference, high viscosity of the continuous phase, or small particle size, centrifuges can be used to increase the rate. They are expensive machines, however, and can rarely be justified except in cases where the value of the product is high and low hold-up in settling devices is required, as in the case of penicillin extraction. Complete separation still requires coalescence, which is not affected by centrifugal force, and a stable emulsion will be merely concentrated and not broken by centrifuging (5). 

Penicillin extraction is an example of the application of process enhancanent via electrostatic fields. Solvent extraction as a means of recovery and purification of penicillin-G and a number of other antibiotics is well-established for these products. Use of liquid-liquid extraction involves operation at close to ambient tanper-ature and equipment-intensive processes such as precipitation and oystallization can be kept to a minimum. The high selectivity offered by chanicaUy suitable solvents has furthered the exploitation of extraction as a recovery ronte. In the case of penicillin-G (pen-G), extraction into a number of solvents is well known and these include n-butyl acetate, amyl acetate, chloroform, and methyl iso-bntyl ketone. 

Figure 8.16 shows the relationship between the mass transfer of product (K a) and applied voltage for penicillin extraction in a 25 mm diameter X 0.65 m height spray column. The importance of electrostatic spraying in the improvement of overall mass transfer rate is clearly demonstrated (Weatherley, 1993). 

Jiang, Y, Xia, H., Yu, J., Guo, C. and Liu, H., Hydrophobic ionic liquids-assisted polymer recovery during penicillin extraction in aqueous two-phase system, Chem. Eng. J. 147, 22-26 (2009). 

There are liquid-liquid extraction systems where the residence time in the device should be very small ( 10 s) to reduce degradation, for example in penicillin extraction carried out at a low temperature. Centrifugal devices (e.g. Podbielniak) are utilized for such systems. As illustrated schematically in Figure 8.1.35(e), this device contains a number of concentric perforated shells around a central shaft rotating rapidly around a central axis. The two liquids are introduced from opposite ends into different tubular channels in the shaft the heavy liquid is introduced near the shaft, whereas the light liquid is introduced near the periphery. 

The behavior of drops in the centrifugal field has been studied (211) and the residence times and mass-transfer rates have been measured (212). PodbieHiiak extractors have been widely used in the pharmaceutical industry, eg, for the extraction of penicillin, and are increasingly used in other fields as weU. Commercial units having throughputs of up to 98 m /h (26,000 gal/h) have been reported. 

Antibiotics. Solvent extraction is an important step in the recovery of many antibiotics (qv) such as penicillin [1406-05-9] streptomycin [57-92-17, novobiocin [303-81-1J, bacitracin [1405-87-4] erythromycin, and the cephalosporins. A good example is in the manufacture of penicillin (242) by a batchwise fermentation. Amyl acetate [628-63-7] or -butyl acetate [123-86-4] is used as the extraction solvent for the filtered fermentation broth. The penicillin is first extracted into the solvent from the broth at pH 2.0 to 2.5 and the extract treated with a buffet solution (pH 6) to obtain a penicillin-rich solution. Then the pH is again lowered and the penicillin is re-extracted into the solvent to yield a pure concentrated solution. Because penicillin degrades rapidly at low pH, it is necessary to perform the initial extraction as rapidly as possible for this reason centrifugal extractors are generally used. 

Isolation. Isolation procedures rely primarily on solubiHty, adsorption, and ionic characteristics of the P-lactam antibiotic to separate it from the large number of other components present in the fermentation mixture. The penicillins ate monobasic catboxyHc acids which lend themselves to solvent extraction techniques (154). Pencillin V, because of its improved acid stabiHty over other penicillins, can be precipitated dkecdy from broth filtrates by addition of dilute sulfuric acid (154,156). The separation process for cephalosporin C is more complex because the amphoteric nature of cephalosporin C precludes dkect extraction into organic solvents. This antibiotic is isolated through the use of a combination of ion-exchange and precipitation procedures (157). The use of neutral, macroporous resins such as XAD-2 or XAD-4, allows for a more rapid elimination of impurities in the initial steps of the isolation (158). The isolation procedure for cephamycin C also involves a series of ion exchange treatments (103). 

MisceUaneous uses include extraction and purification of penicillin, alkaloids, vitamins, and flavors, and as an intermediate in the preparation of dyes and pesticides. Chloroform has also been used as a fumigant and insecticide, in the formulation of cough symps, toothpastes, liniments, and toothache preparations. These latter uses were banned by the FDA in 1976 (38). 

The pH of the mixture was adjusted to 7.5 by adding a saturated sodium bicarbonate solution. After being washed twice with diethyl ether, the reaction solution was acidified to pH 2 with dilute hydrochloric acid and extracted with ether. The ether solution containing the free penicillin was washed twice with water and then extracted with 50 ml of N potassium bicarbonate solution. After freeze drying of the obtained neutral solution, the potassium salt of o-azidobenzylpenicillin was obtained as a slightly colored powder (11.2 grams, 54% yield) with a purity of 55% as determined by the hydroxylamine method (the potassium salt of penicillin G being used as a standard). 

There was added to 250 ml of a concentrated butyl acetate extract containing 74,000 units of the acid form of penicillin per ml, 50 ml of a butyl acetate solution containing 0.238 g per ml of procaine base. The solution was agitated for one hour. The precipitate which formed was very gummy and not in the form of discrete crystals. This precipitate was crystallized by scratching the side of the vessel and agitating further. After this treatment 18.25 g of crystalline procaine penicillin was obtained which assayed 1010 units per mg representing a yield of 99.6% of the activity contained in the concentrated extract. 

Biotechnological processes may be divided into fermentation processes and biotransformations. In a fermentation process, products are formed from components in the fermentation broth, as primary or secondary metabolites, by microorganisms or higher cells. Product examples are amino acids, vitamins, or antibiotics such as penicillin or cephalosporin. In these cases, co-solvents are sometimes used for in situ product extraction. 

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