10 Approaches to Cephalosporin C Purification from Fermentation Broth
Downloaded by UNIV LAVAL on October 19, 2015 | http://pubs.acs.org Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch010
M. E. WILDFEUER Eli Lilly and Company, Lafayette, IN 47902
Different antibiotics generally lend themselves to specific purification techniques. Ceph C, however, is fairly unique since i t s separation from broth can be carried out by a variety of methods, each of which illustrates a particular approach to antibiotic isolation. Part of the reason for this versatile behavior results from the fact that ceph C is not the end use product, and for this reason modifications to the molecule to facilitate i t s separation or alter its physical characteristics—without affecting the active part of the ceph C molecule—are possible. Examples of techniques used for purification of ceph C or i t s derivatives include: (1) carbon adsorption, (2) non-ionic resin adsorption, (3) ion exchange, (4) solvent extraction of derivatives, (5) precipitation of derivative acid or salt, (6) precipitation of metal salt, (7) broth drydown, (8) azeotropic extraction, and (9) enzymic modification. Many of these methods are not merely laboratory curiosities but have been successfully applied to large scale production of ceph C. The from used • • • • • • • •
initial isolation of antibiotics and other fermentation products fermentation broths can take many routes. Those that have been on a large scale have included the following: Carbon adsorption Non-ionic resin adsorption Ion-exchange adsorption (anion or cation) Solvent extraction Precipitation Broth drydown Azeotropic extraction Enzymic modification 0097-6156/85/0271-0155S06.00/0 © 1985 American Chemical Society In Purification of Fermentation Products; LeRoith, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.
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A l t h o u g h one o r t h e o t h e r o f these t e c h n i q u e s has t y p i c a l l y been a p p l i e d t o a p a r t i c u l a r a n t i b i o t i c o r c l a s s of a n t i b i o t i c s , c e p h a l o s p o r i n C i s r a t h e r unique i n t h a t each of t h e s e methods c a n be u t i l i z e d s u c c e s s f u l l y f o r i t s i s o l a t i o n . To g e t some p e r s p e c t i v e on p u r i f i c a t i o n c o m p l e x i t i e s , T a b l e I l i s t s some o f the major components found i n a f e r m e n t a t i o n b r o t h .
Downloaded by UNIV LAVAL on October 19, 2015 | http://pubs.acs.org Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch010
Table I . B r o t h I m p u r i t i e s Classification
Examples
I n s o l u b l e Components
Mycelia N o n - u t i l i z e d raw m a t e r i a l s Insoluble s a l t s Immiscible o i l s Insoluble metabolites S o l u b l e n o n - u t i l i z e d raw m a t e r i a l s Soluble metabolites P o l y s a c c h a r i d e s and sugars P r o t e i n s and amino a c i d s Lipids Nucleic acids "Unique m e t a b o l i t e s
S o l u b l e Components
11
The m y c e l i a and o t h e r i n s o l u b l e substances a r e u s u a l l y removed by a f i l t r a t i o n s t e p , a l t h o u g h whole b r o t h r e s i n o r s o l v e n t ext r a c t i o n methods may be used. I t s h o u l d be noted t h a t c e p h a l o s p o r i n C i s s o l u b l e i n f e r m e n t a t i o n b r o t h (some a n t i b i o t i c s a r e n o t ) . The magnitude o f t h e i s o l a t i o n c h a l l e n g e i s i l l u s t r a t e d by t h e f a c t t h a t 20-70 m i l l i o n l i t e r s o f b r o t h may t y p i c a l l y be h a r v e s t e d a n n u a l l y . Systems capable o f h a n d l i n g b r o t h f l o w r a t e s o f 100-200 l i t e r s per minute might be r e q u i r e d . The s t r u c t u r e o f c e p h a l o s p o r i n C, a (3-lactam a n t i b i o t i c , i s shown i n F i g u r e 1. Under n e u t r a l , b u t e s p e c i a l l y b a s i c c o n d i t i o n s , i t i s h y d r o l y z e d t o d e s a c e t y l c e p h a l o s p o r i n C. I n a c i d c e p h a l o s p o r i n C l a c t o n e i s formed (1,2) (both o f t h e s e a r e shown i n F i g u r e 1 ) . I n o r d e r t o m i n i m i z e these d e g r a d a t i o n s , i t i s i m p o r t a n t t h a t c e p h a l o s p o r i n C b r o t h be p r o c e s s e d r a p i d l y , a v o i d i n g extremes o f pH and k e e p i n g temperatures low. A c t i v a t e d Carbon A c t i v a t e d carbon w i l l e f f e c t i v e l y remove c e p h a l o s p o r i n C from b r o t h ( 3 , 4 ) ; e l u t i o n i s e f f e c t e d w i t h d i l u t e aqueous s o l v e n t s . The c a r b o n column e l u a t e may then be p u r i f i e d f u r t h e r by a d s o r p t i o n and e l u t i o n of c e p h a l o s p o r i n C u s i n g an a n i o n exchange r e s i n , s i n c e most of t h e competing s t r o n g a n i o n s a r e n o t adsorbed t o carbon. An example of a c a r b o n - a n i o n exchange r o u t e i s seen i n F i g u r e 2. One o f t h e d i f f i c u l t i e s o f t h i s method i s t h e requirement t h a t carbon be p e r i o d i c a l l y r e a c t i v a t e d or r e p l a c e d . A l s o , as mentioned, e l u t i o n and r e g e n e r a t i o n r e q u i r e s o l v e n t , and c a p a c i t y f o r c e p h a l o s p o r i n C i s r e l a t i v e l y low.
In Purification of Fermentation Products; LeRoith, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.
Ceph C Purification from Fermentation Broth
WILDFEUER
NH -CH ( C H ^ C O N H - j — ^ V 2
COOH
C
0
Ceph C
H
^ T ^ 2 ° COOH
C
O
C
H
3
pH
