Cephalosporin broth

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). 

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. 

After a strain improvement and development programme similar to, but more complicated than that of penicillin, the D-a-aminoadipyl side chain containing cephalosporin C was obtained by large scale fermentation. However, cephalosporin C could not be isolated as easily as penicillin G or V. Due to its amphoteric nature it is soluble at any pH in the fermentation broth. Several costly isolation procedures involving ion-exchange chromatography have been developed, as a result of which cephalosporin C is much more expensive than penicillin G. 

The system was applied to the determination of cephalosporin c in a broth of Cephalospolium acremonium, and was compared with a method based on high-pressure liquid chromatography (h.p.l.c.). 

Separation of liquid olefin/paraffin mixtures Removal of 2-chlorophenol Ethanol removal from aqueous solutions Separation of cephalosporin C from fermentation broth Separation of penicillin G from aqueous streams Enrichment of amino acids... 

SCHEME 2. Process of the extraction of a Cephalosporin C derivative from fermentation broth. 

One such advance was the work carried out in Sefton Park by Dr. Robert Fildes and co-workers5,6 on the enzyme-mediated conversion of the zwitterionic aminoadipic acid side chain in Cephalosporin C into a glutaroyl side chain (a process that rendered the molecule more amenable to solvent extraction from filtered fermentation broths ... 

SCHEME 2. Antibioticos industrial process for the conversion of Cephalosporin C in fermentation broths to 7-ACA. 

Sahoo GC, Dutta NN, and Dass NN. Liquid membrane extraction of cephalosporin-C from fermentation broth. J Mem Sci, 1999 157(2) 251-261. 

Figure 12.18 Purification of cephalosporin C from a fermentation broth. (Left) Analysis. Same experimental conditions as in displacement. (Right) Displacement. Column 350 x 4.6 mm, packed with 5 im Zorbax carrier 1% v/v acetonitrile in 20 mM sodium acetate pH 5.2 displacer 40 mg/mL BEE in carrier feed 5 mL fermentation broth flow rate 1.0 mL/min fraction volume 300 jiL. Reproduced with permission from G. Subramanian, M. Phillips and S. Cramer,. Chromatogr., 439 (1988) 341 (Fig. 5).

Approaches to Cephalosporin C Purification from Fermentation Broth... 

The mycelia and other insoluble substances are usually removed by a filtration step, although whole broth resin or solvent extraction methods may be used. It should be noted that cephalosporin C is soluble in fermentation broth (some antibiotics are not). The magnitude of the isolation challenge is illustrated by the fact that 20-70 million liters of broth may typically be harvested annually. Systems capable of handling broth flow rates of 100-200 liters per minute might be required. 

The structure of cephalosporin C, a 3-lactam antibiotic, is shown in Figure 1. Under neutral, but especially basic conditions, it is hydrolyzed to desacetyl cephalosporin C. In acid cephalosporin C lactone is formed (1,2) (both of these are shown in Figure 1). In order to minimize these degradations, it is important that cephalosporin C broth be processed rapidly, avoiding extremes of pH and keeping temperatures low. 

Activated carbon will effectively remove cephalosporin C from broth (3,4) elution is effected with dilute aqueous solvents. The carbon column eluate may then be purified further by adsorption and elution of cephalosporin C using an anion exchange resin, since most of the competing strong anions are not adsorbed to carbon. An example of a carbon-anion exchange route is seen in Figure 2. 

Most of the derivatives shown in Figure 8 are solvent extractable at low pH, and thus one of the classical methods used for antibiotic purification becomes accessible to cephalosporin C. To be commercially feasible, solvents should be selective and only slightly miscible with water. Extraction efficiency should be sufficiently high that multiple extractions are not required, and ideally should be efficient at low ratios so as to effect a concentration of the desired component. Emulsions and insoluble solids are anathema to extraction. Using these criteria, extraction of most of the cephalosporin C derivatives at low pH are far from ideal since mostly non-selective solvents (such as n-butanol and ethyl acetate) usually work best several extractions seem to be required, and derivatized cephalosporin C broth upon acidification will frequently result in emulsion formation. However, some derivatives behave better than... 

Although of itself broth drydown is not a very significant purification step, it can offer certain advantages. As the example in Figure 10 shows, the dry product can be reslurried in another solvent (in this case acetic acid), the cephalosporin C derivatized (with acetic anhydride) and finally isolated as the zinc salt of N-acetyl cephalosporin C (46). 

Figure 10. Broth drydown and azeotropic extraction for cephalosporin C isolation.

Acetoxy displacement in broth prior to isolation of cephalosporin C (6,21,31,37,48-50) is compared schematically in Figure 12 with the more usual approach to cephalosporin C purification. Derivatization at C-3 can be used in conjunction with other methods such as non-ionic resin adsorption or N-derivatized extraction. However, no advantage in terms of reduced solubility or enhanced extractability is indicated for these derivatives. This alternative, however, does effectively eliminate one step in the chain and could result in an overall yield benefit. 

No discussion of cephalosporin C isolation would be complete without some comment on desacetyl cephalosporin C. This compound, always present in significant quantity in broth, besides being a neutral and high pH degradation product of cephalosporin C (2,61), is also produced enzymatically by endogenous or contaminant esterases. 

There is, of course, no one "right" way to process cephalosporin C broth. It is a tribute to the purification chemists and engineers that so many innovative approaches have been developed for cephalosporin C isolation. A decision to choose a particular route generally involves weighing the following areas ... 

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