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9 Process Developments in the Isolation of Largomycin F-II, a Chromoprotein Antitumor Antibiotic Downloaded by UNIV OF QUEENSLAND on October 20, 2015 | http://pubs.acs.org Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch009

RAMESH C. PANDEY1, CHABI C. KALITA2, MARK E. GUSTAFSON3, MARGARET C. KLINE, MICHAEL E. LEIDHECKER4, and JOHN T. ROSS National Cancer Institute, Frederick Cancer Research Facility, Fermentation Program, Frederick, MD 21701

Processes for the large scale isolation and p u r i f i cation of biologically active largomycin F-II, a chromoprotein antitumor antibiotic, produced by Streptomyces pluricolorescens MCRL-0367, from filtered fermentation broth and from mycelium ( c e l l paste) are described. Advantages of the more recent filtration/concentration units over the earlier methods, in process simplification, are discussed. These processes differ primarily in the initial steps used to recover and concentrate the material. In all cases, gel f i l t r a t i o n on Sephadex G—100 and hydroxylapatite are used in the final stages of purification to yield gram quantities of purified biologically active largomycin F-II.

The isolation of largomycin F-II, a chromoprotein antitumor anti biotic, from the culture f i l t r a t e of Streptomyces ρluricolores cens MCRL-0367 (1-3) was reported i n 1970. It was shown to have biologi cal activity against several tumors, including KB, Ρ388, HeLa S-3 cells (0.1 yg/ml), Ehrlich ascites carcinoma, Sarcoma-180 and SN-36 leukemia (3). Some of i t s physicochemical and biological proper ties are summarized in Table I , and the partial amino acids sequence is shown i n Figure 1 (4). Because of i t s biological activity, largomycin F-II was selected by the National Cancer Institute (NCI) for further formulation, toxicological and possible c l i n i c a l studies, a situation requiring gram quantities of pharmaceutical grade material. Current Current 3 Current 4 Current 1

2

address: Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ 08854. address: 10919 Rawley Road, Mount Airy, MD 21771. address: Monsanto Company, St. Louis, MO 63167. address: 190 Stone Gate Drive, Frederick, MD 21701. 0097-6156/85/0271-0133$06.00/0 © 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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Downloaded by UNIV OF QUEENSLAND on October 20, 2015 | http://pubs.acs.org Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch009

Table I . P h y s i c o c h e m i c a l and B i o l o g i c a l P r o p e r t i e s of Largomycin F - I I

P r o d u c i n g organism (JL) Nature ( 2 ) M o l e c u l a r weight ( 4 )

Streptomyces

I s o e l e c t r i c point (4^

4.13

1% UV λ max ( Ε χ ' ^ ) ( 4 ) i n 0.1 Ν HC1 i n 0.1 Ν NaOH A n t i b i o t i c a c t i v i t y (_3) Antitumor a c t i v i t y ( 3 )

pluricolores

cens

MCRL-0367

Acidic, 30,000, 58,000, 29,300,

y e l l o w amorphous powder gel electrophoresis u l t r a c e n t r i f u g a t i o n i n PBS u l t r a c e n t r i f u g a t i o n i n 6M guanidine hydrochloride (pH g r a d i a n t 3.5-5.0)

272 ( 1 6 . 4 ) , 420 nm ( 1 . 8 ) 288 s h ( 1 5 . 6 ) , 525 nm ( 2 . 0 ) A c t i v e a g a i n s t S. aureus 5. lut eau A c t i v e a g a i n s t KB, P388, HeLa, Ehrlich ascites, Sarcoma-180 and SN-36 leukemia

P r e l i m i n a r y s c a l e - u p assessments n e c e s s i t a t e d t h e i n v e s t i g a t i o n of t h e f o l l o w i n g c r i t e r i a t o a c h i e v e t h i s g o a l : 1.

2. 3.

4.

S i n c e t i t e r s of t h e a n t i b i o t i c i n t h e p r o d u c i n g s t r a i n a r e g e n e r a l l y low - s t r a i n and f e r m e n t a t i o n development work i s necessary. The development of assay methods i s r e q u i r e d s i n c e q u a n t i t a t i v e assay methods a r e n o t e s t a b l i s h e d . P r o c e s s e s f o r t h e l a r g e s c a l e i s o l a t i o n and p u r i f i c a t i o n a r e as y e t n o t w e l l e s t a b l i s h e d - t h e r e f o r e p r o c e s s development work i s necessary. As t h e m a t e r i a l i s d e s t i n e d f o r phase I c l i n i c a l t r i a l s , i t must conform t o GMP, GLP and FDA r e g u l a t i o n s .

A l l f o u r p o i n t s have been addressed by v a r i o u s groups i n t h e NCI-FCRF F e r m e n t a t i o n Program. T h i s paper w i l l concern i t s e l f p r i m a r i l y w i t h p r o c e s s development work f o r t h e p u r i f i c a t i o n of gram q u a n t i t i e s of l a r g o m y c i n F - I I . As work i n one a r e a impacts on t h e o t h e r s , v a r i o u s a s p e c t s of a l l f o u r p o i n t s w i l l a l s o be d i s c u s s e d where a p p r o p r i a t e . M a t e r i a l s And Methods G e n e r a l Comments. A l l h i g h performance l i q u i d chromatography (HPLC) a n a l y s e s were c a r r i e d out on a Waters Assoc. WISP 710B automatic i n j e c t o r and a S c h o e f f e l SF770 S p e c t r o f l o w v a r i a b l e - w a v e l e n g t h UV-Vis d e t e c t o r . The d e t e c t o r was s e t a t 210 nm and 0.04 a . u . f . s . ( c e l l volume 8 μΐ; path l e n g t h 10 mm). Waters A s s o c . p r o t e i n a n a l y s i s column 1-125 (7.8 mm χ 300 mm) and a m o b i l phase c o n s i s t i n g o f 0.05 M Na HP04/NaH P04, pH 6.8 a t a f l o w r a t e of 1 ml/min and a c h a r t speed of 0.5 cm/min was used f o r a l l t h e HPLC work. 2

2

In Purification of Fermentation Products; LeRoith, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

9.

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Isolation of Largomycin F-II

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Sephadex G-100 ( p a r t i c l e s i z e 40 ^ 120 μ) purchased from P h a r macia F i n e C h e m i c a l s , D i v i s i o n of P h a r m a c i a , I n c . , P i s c a t a w a y , NJ 08854, was used f o r a l l Sephadex column chromatography. Diethylarainoethyl c e l l u l o s e (DE-23) f i b r o u s form, purchased from Whatman, I n c . , C l i f t o n , NJ 07014, was used f o r a l l DE-23 column chromatogra phy. U l t r a f i l t r a t i o n and d i a f i l t r a t i o n were performed u s i n g Amicon DC-50 and DC-4 h o l l o w f i b e r systems and/or a M i l l i p o r e P e l l i c o n u n i t , depending on s c a l e of o p e r a t i o n . A v a r i e t y of pumps were used throughout the l a r g o m y c i n F - l l p r o d u c t i o n campaign, i n c l u d i n g p e r i s t a l t i c , c e n t r i f u g a l , s u r g e , as w e l l as p o s i t i v e - d i s p l a c e m e n t gear. L a r g o m y c i n F - I I A s s a y s . We have adopted the HPLC method of a n a l y s i s f o r r o u t i n e l a r g o m y c i n F - I I a s s a y , because i t i s s i m p l e , r a p i d (y 15 m i n / a s s a y ) , q u a n t i t a t i v e , does not r e q u i r e e x t e n s i v e sample p r e p a r a t i o n and i s s e l e c t i v e i n s e p a r a t i n g m i x t u r e s on the b a s i s o f m o l e c u l a r w e i g h t . However, t h i s method cannot d i s t i n g u i s h b i o l o g i c a l l y i n a c t i v e p r o t e i n s from a c t i v e p r o t e i n s , n o r c o u l d i t r e s o l v e F - I I from s e v e r a l p r o t e a s e s produced i n t h e f e r m e n t a t i o n and of a p p r o x i m a t e l y the same m o l e c u l a r weight as F - I I . C l a s s i c a l r e v e r s e phase g r a d i e n t HPLC systems were a l s o not u s e f u l , because t h e a c i d i c n a t u r e of the s o l v e n t r o u t i n e l y used (0.05% TFA) r e s u l t e d i n i n a c t i v a t i o n of F - I I . Due t o t h e s e d e f i c i e n c i e s HPLC assay i s used i n c o n j u n c t i o n w i t h two measures of b i o l o g i c a l a c t i v i t y , the B i o c h e m i c a l I n d u c t Assay (BIA) (5,6) and Micrococcus luteus (ML) a s s a y s t o f o l l o w the p r o d u c t i o n and i s o l a t i o n of l a r g o m y c i n F - I I . P r e p a r a t i o n of Samples f o r HPLC Assay. From B r o t h . The b r o t h sample i s c e n t r i f u g e d and the s u p e r n a t a n t f i l t e r e d t h r o u g h a M i l l i p o r e (0.45) f i l t e r . The f i l t r a t e i s i n j e c t e d d i r e c t l y onto the HPLC column. A HPLC p r o f i l e of such an i n j e c t i o n i s shown i n F i g u r e 2. From M y c e l i u m . M y c e l i a a r e suspended i n phosphate b u f f e r (0.05 M, pH 6.8, 5 ml/g) and s o n i c a t e d f o r 5 min i n a s o n i c water b a t h p r i o r t o c e n t r i f u g a t i o n . A f t e r c e n t r i f u g a t i o n the e x t r a c t i s p r o c e s s e d i n a manner s i m i l a r t o b r o t h sample. A HPLC p r o f i l e of m y c e l i a e x t r a c t i s shown i n F i g u r e 2. B i o c h e m i c a l I n d u c t Assay (BIA) (_5 ,J&). The s e m i - q u a n t i t a t i v e spot t e s t v e r s i o n ( n o t the one-tube assay f o r the q u a n t i t a t i v e measurement of i n d u c t i o n ) was used f o r most of the work. The bac t e r i a a r e poured i n agar w i t h o r w i t h o u t r a t l i v e r S9 a c t i v a t i o n mix, onto l a r g e (24 cm χ 24 cm) b i o a s s a y p l a t e s . L a r g o m y c i n f e r m e n t a t i o n b r o t h o r t e s t s o l u t i o n s a r e s p o t t e d onto the p l a t e s , a l l o w e d t o i n c u b a t e f o r t h r e e hours a t 37°C, and o v e r l a i d w i t h a second agar l a y e r c o n t a i n i n g s u b s t r a t e . W i t h i n f i v e o r t e n m i n u t e s , a r e a s of i n d u c t i o n a r e seen as red s p o t s of i n s o l u b l e dye formed by c l e a v a g e of the c o l o r l e s s s u b s t r a t e . R a p i d s a m p l i n g w i t h the wide end of p a s t e u r p i p e t t e s a l l o w s an o p e r a t o r t o s p o t 100 samples on two p l a t e s i n 20 m i n u t e s . Micrococcus luteus (ATCC 9341) A s s a y . M u e l l e r - H i n t o n agar p l a t e s were o v e r l a y e d w i t h 0.7% D i f c o B a c t o - a g a r c o n t a i n i n g a p p r o x i m a t e l y 2.5 χ 10^ c e l l s o f M. luteus/ml. When the agar i s s e t , 15 μ 1 of


136


1 5 10 Asp-lle-Leu-lle-Ala-Gly-Ala-Thr-Gly-Asn11 15 20 Val-Gly-Lys-Pro-Leu-Val-Glu-Gly-Leu-Leu21 25 30 Ala-Ala-Gly-Lys-Pro-Val-Arg-Ala-Leu-Thr-Arg-Asn—


-Ala-Ala-Leu-Phe-OH F i g u r e 1. Proposed p a r t i a l s t r u c t u r e f o r a p o p r o t e i n o f l a r g o m y c i n F-II. ( 4 ) .

III III ι nm

I II III

ttt

ttt

tt

I II III ttt

0

HI

Pellet

Supernatant

Pellet

Supernatant

60 Hrs.

96 Hrs.

72 Hrs.

120 Hrs.

F i g u r e 2. High performance l i q u i d chromatography p r o f i l e of c e n t r i f u g e d whole b r o t h and mycelium e x t r a c t . Column: Waters Assoc. 1-125 p r o t e i n a n a l y s i s column (30 cm χ 7.8 mm). M o b i l e phase: 0.05 M phosphate b u f f e r , pH 6.9. Flow r a t e : 1.0 ml/min, D e t e c t o r : UV a t 210 nm.


9. PANDEY ET AL.


137


sample o r s t a n d a r d i s s p o t t e d onto 6 mm paper d i s c s , and the d i s c s a r e t r a n s f e r r e d wet onto the agar p l a t e , and the p l a t e i s i n c u b a t e d o v e r n i g h t a t 37°C. A f t e r 20-24 h o u r s , zones are measured i n mm, and the l o g conc e n t r a t i o n of s t a n d a r d i s p l o t t e d a g a i n s t zone s i z e i n mm. MIC a r e determined by e x t r a p o l a t i o n of the s t a n d a r d curve t o the Y - a x i s . The c o n c e n t r a t i o n of l a r g o m y c i n F - I I i n samples i s determined by measuring zone s i z e i n mm and r e a d i n g the c o n c e n t r a t i o n o f f the standard curve. Protease Assay. N o n s p e c i f i c protease a c t i v i t y i n largomycin F - I I was determined by A z o c o l l a s s a y . V a r y i n g amounts of l a r g o m y c i n F - I I (20 yg * 100/yg) d i s s o l v e d i n 20 mM T r i s - H C l (pH 7.2) were taken i n s t o p p e r e d t u b e s . The same b u f f e r was added i n each tube t o make the volume t o 100 y l . To each tube 100 y l of a z o c o l l (100 mg/5 ml w a t e r ) was added and i n c u b a t e d a t 37°C f o r 1 hour. The r e a c t i o n was t e r m i n a t e d by adding 2 ml of 5% t r i c h l o r o a c e t i c a c i d t o each tube. The tubes were c e n t r i f u g e d f o r 15 minutes a t 25,000 rpm and the o p t i c a l d e n s i t y of the s u p e r n a t a n t was measured a t 366 nm. I t was found t h a t the measured p r o t e a s e a c t i v i t i e s were l i n e a r l y p r o p o r t i o n a l t o the amount of enzyme. R e s u l t s And D i s c u s s i o n I s o l a t i o n of Largomycin F - I I . F i g u r e 3 g r a p h i c a l l y i l l u s t r a t e s HPLC t i t e r s vs c u l t u r e age f o r both c e l l f r e e b r o t h and m y c e l i a l e x t r a c t s f o r a t y p i c a l 1 4 - l i t e r and 3 0 0 - l i t e r f e r m e n t a t i o n s . P r i o r t o a t t e m p t i n g any p r o c e s s development work, i t was d e c i d e d t o e v a l u a t e the p r o c e s s r e p o r t e d by Yamaguchi et al. (_2) f o r the i s o l a t i o n of l a r g o m y c i n F - I I . P r o c e s s of Yamaguchi et al.

(2)

I n t h i s p r o c e s s (Scheme 1) the l a r g o m y c i n complex was s e p a r a t e d i n t o t h r e e components F - I , F - I I and F - I I I by a p p l y i n g the f o l l o w i n g procedure i n s u c c e s s i o n : (A) g r a d i e n t e x t r a c t i o n w i t h aqueous ammonium s u l f a t e s o l u t i o n , (B) i s o e l e c t r i c p o i n t p r e c i p i t a t i o n , and (C) s i z e e x c l u s i o n g e l f i l t r a t i o n on Sephadex G-100. T h i s procedure y i e l d e d l a r g o m y c i n F - I I t h a t possessed l i t t l e b i o l o g i c a l a c t i v i t y , was impure on SDS p o l y a c r y l a m i d e g e l s and cont a i n e d p r o t e a s e a c t i v i t i e s . I n a d d i t i o n the procedure r e s u l t e d i n a low y i e l d , r e s u l t i n g ca. 1.5 grams of m a t e r i a l from a 2000 L f e r mentation. I t has been observed and r e p o r t e d i n the l i t e r a t u r e t h a t f u r t h e r p u r i f i c a t i o n of low s p e c i f i c a c t i v i t y F - I I d i d not h e l p i n i n c r e a s i n g the b i o l o g i c a l a c t i v i t y of the m a t e r i a l . A t t h i s s t a g e , then the f o l l o w i n g p o i n t s were c o n s i d e r e d : 1.

2.

Largomycin F - I I i s o n l y one of many " l a r g o m y c i n s " of s i m i l a r s t r u c t u r e and b i o a c t i v i t y . Largomycin F - I I i s not n e c e s s a r i l y the most b i o l o g i c a l l y a c t i v e of these components. Largomycin F - I I i s a c h r o m o p r o t e i n . L i k e the p r o t e i n a n t i t u m o r a n t i b i o t i c s n e o c a r z i n o s t a t i n (7j-9) * macromomycin ( 10-12), t h e chromophore i s p r o b a b l y r e s p o n s i b l e f o r the b i o l o g i c a l a c t i v i t y . a n c



138


Time (Hrs.)

F i g u r e 3. Time course o f l a r g o m y c i n F - I I p r o d u c t i o n i n 14 l i t e r ( l e f t ) and 300 l i t e r ( r i g h t ) t a n k s .


9.

PANDEY ET AL.


139

Crude Largomycin Complex

(Steps 1-3)

605g Extracted with 0.7 satd (NH ) S0 , 1.0L Filtered Step-4 Residue 4 2

4

J Wash with 0.7 satd (NH ) S0 , 0.5L 4 2

4

Residue Extracted with 0.5 satd (NH ) S 0 1.2L Filtered Step-5 Residue Wash with 0.5 satd (NH ) S0 , 0.8L 4 2

Filtrate, 1.2L


f

4

2

4t

4

Wash, 0.8L Residue (F-l) Extracted with water Filtered Step-6

Combined Precipitated with Solid (NH ) S0 , 500g Added celite, 40g, stirred 10h Filtered 4

2

nitrate Dialyzed Lyophilized

4

Largomycin F-l (2.4 g)

Residue (F-ll and F-lll)

Residue (F-ll and F-lll) ' Exl Extracted with 0.7 satd (NH ) S0 , 1.0L Fill Filtered Step-7 4 2

4

Residue I Extracted with 0.53 satd (NH ) S0 , 1.5L Filtered Step-8 4 2

4

Filtrate, 1.44L Precipitated with solid (NH ) S 0 360 g Added Celite, Stirred 10h Filtered Step-9 4 2

4l

Residue Extracted with (1:1) mixture of satd (NH ) S0 ard 0.1 M acetate buffer (pH 4.0), 0.5L Filtered Step-10 4 2

4

Filtrate, 0.5L Dialyzed Lyophilized

Residue Extracted with (1:1) mixture of 0.1 M acetate buffer pH6.0 and satd (NH ) S0 , 0.5L Filtered

Step-11

Filtrate (0.5L) Dialyzed Lyophilized

Step-12

4

Brown product Largomycin F-lll (31 mg)

2

4

Yellow white product Largomycin F-ll (40 mg)

Scheme 1. P u r i f i c a t i o n of l a r g o m y c i n F - I I by the p r o c e s s of Yamaguchi e t a l . (2)



140


3. As a c h r o m o p r o t e i n , l a r g o m y c i n i s p r o b a b l y l i g h t s e n s i t i v e . Exposure t o l i g h t r e s u l t s i n an i n a c t i v e p r o t e i n w i t h s i m i l a r , i f not i d e n t i c a l p h y s i c o c h e m i c a l p r o p e r t i e s . 4. D u r i n g the f e r m e n t a t i o n , chromophore and p r o t e i n a r e p r o b a b l y produced s e p a r a t e l y , and o n l y p a r t of the chromophore i s a t t a c h e d t o the l a r g o m y c i n F - I I p r o t e i n . The e x i s t e n c e of f r e e chromophore e x p l a i n s the appearance of b i o l o g i c a l a c t i v i t y i n a f e r m e n t a t i o n b r o t h b e f o r e the appearance of l a r g o m y c i n F - I I , and why t o t a l b i o a c t i v i t y does not n e c e s s a r i l y c o r r e l a t e w i t h t o t a l amount of F - I I . 5. S e v e r a l p r o t e a s e s are c o - p u r i f i e d w i t h l a r g o m y c i n F - I I which i n s o l u t i o n degrade the l a r g o m y c i n F - I I p r o t e i n . 6. I t has been p o s s i b l e t o i s o l a t e the chromophore and the p r o t e i n under a c i d i c pH w i t h a s u i t a b l e s o l v e n t . A l l these o b s e r v a t i o n s are summarized i n a non-chemical r e p r e s e n t a t i o n form i n F i g u r e 4. The l a n t e r n s w i t h l i g h t r e p r e s e n t the a c t i v e chromophore, the persons r e p r e s e n t the p r o t e i n , the dark l a n t e r n s r e p r e s e n t the i n a c t i v e chromophore. The e f f e c t of p r o t e a s e s i s shown by the breakage of the l i m b , head or body. The v a r i o u s diagrams i n a c i r c l e or box i n d i c a t e what can be i s o l a t e d by u s i n g a s u i t a b l e procedure i n a p a r t i c u l a r s t e p . The p r i m a r y drawback of the Yamaguchi, et al. p r o c e s s (Scheme 1) was the l a r g e volume of the f e r m e n t a t i o n b r o t h and the amount of ammonium s u l f a t e needed. S i n c e a number of new t e c h n o l o g i e s have e v o l v e d i n the p r o t e i n p u r i f i c a t i o n d u r i n g the l a s t decade, i t was f e l t t h a t a more e f f i c i e n t a l t e r n a t i v e p r o c e s s c o u l d be developed t o i s o l a t e and p u r i f y the b i o l o g i c a l l y a c t i v e l a r g o m y c i n F - I I . With these p o i n t s i n mind and the v a r i o u s equipment a t hand, a l t e r n a t i v e p r o c e s s e s were developed f o r the p u r i f i c a t i o n of l a r g o m y c i n F - I I u s i n g the t e c h n i q u e s of d i a f i l t r a t i o n , u l t r a f i l t r a t i o n , i o n exchange chromatography, g e l permeation chromatography, h y d r o x y l a p a t i t e column chromatography and l y o p h i l i z a t i o n . NCI-FCRF P r o c e s s I (Scheme 2 ) . I n t h i s p r o c e s s f i l t e r e d or c e n t r i fuged b r o t h i s used as the s t a r t i n g m a t e r i a l f o r the i s o l a t i o n of l a r g o m y c i n F - I I . The f i l t r a t e was d i a l y z e d a g a i n s t d e i o n i z e d water t o remove low m o l e c u l a r weight m a t e r i a l , and c o n c e n t r a t e d i n an Amicon DC-50 h o l l o w f i b e r u l t r a f i l t r a t i o n u n i t equipped w i t h 10 Κ cut o f f h o l l o w f i b e r s . A f t e r the i n i t i a l u l t r a f i l t r a t i o n concen t r a t i o n , advantage i s t a k e n of the e a r l i e r work t h a t l a r g o m y c i n F - I I s t a y s i n s o l u t i o n i n 0.5 s a t u r a t e d ammonium s u l f a t e s o l u t i o n , t h e r e f o r e an e q u a l volume of s a t u r a t e d ammonium s u l f a t e s o l u t i o n was added t o the c o n c e n t r a t e and l e t s t a n d at 4°C f o r 24 h o u r s . [Con c e n t r a t i o n f i r s t reduces the amount of ammonium s u l f a t e needed, speeds the p r o c e s s , and makes p r e c i p i t a t i o n and i t s r e c o v e r y f a s t e r and more e f f i c i e n t . ] The c o n c e n t r a t e i s then f i l t e r e d , d i a l y z e d , and c o n c e n t r a t e d through 10 Κ Amicon h o l l o w f i b e r s . Largomycin F - I I was then f u r t h e r p u r i f i e d by passage through Sephadex G-100 or DE-23 a n i o n exchange c e l l u l o s e columns. Both chromatographic p r o c e d u r e s , e i t h e r a l o n e or i n c o n j u n c t i o n w i t h the o t h e r , gave m a t e r i a l of comparable q u a l i t y . The l a r g o m y c i n F - I I r i c h f r a c t i o n s , as judged by HPLC and b i o a c t i v i t y were combined, d i a l y z e d , c o n c e n t r a t e d and l y o p h i l i z e d t o a l i g h t y e l l o w powder. The e n t i r e p u r i f i c a t i o n scheme, t o g e t h e r w i t h the HPLC t r a c i n g s of the m a t e r i a l a t v a r i o u s s t a g e s , i s shown i n Scheme 2.



Figure 4. Non-chemical r e p r e s e n t a t i o n of the p r o d u c t i o n and i s o l a t i o n of the chromoprotein a n t i t u m o r a n t i b i o t i c , l a r g o m y c i n F-II. The person w i t h a l i g h t e d l a n t e r n i s the b i o l o g i c a l l y a c t i v e form of l a r g o m y c i n F - I I .



142

Whole Fermentation Broth I Centrifuge/Filter


Residue (Discard)

Filtered Broth Dialysis/concentration to - 1/10th volume

Dialysate/concentrate out (Discard) Concentrate Add equal vol. of satd. Ammonium Sulfate solution. Let stand at 4 /24 hr. Filter through celite e

Residue (Discard)

Filtrate Dialysis/concentration to - 1/5th volume

Dialysate/concentrate out (Discard) Concentrate JDEAE Column Largomycin F-ll Fractions I Dialysis/concentration Dialysate/concentrate out (Discard)

Concentrate |Lyophilization Largomycin F-ll Powder

L

JL-

Scheme 2. P u r i f i c a t i o n o f l a r g o m y c i n F - I I from f i l t e r e d b r o t h u s i n g r e c e n t methods (NCI-FCRF P r o c e s s I ) . HPLC p a t t e r n of t h e l a r g o m y c i n p u r i t y i s shown on the s i d e .


9.

PANDEY ET AL.


143


The l a r g o m y c i n F - I I i s o l a t e d i n t h i s manner s t i l l c o n t a i n e d a p p r e c i a b l e p r o t e a s e a c t i v i t y and showed i m p u r i t y on SDS g e l s . I t was a l s o noted a t t h i s time t h a t i f the f e r m e n t a t i o n i s t e r m i n a t e d at about 48 h o u r s , most of the l a r g o m y c i n F - I I remains a s s o c i a t e d w i t h the mycelium ( F i g u r e 3). M a t e r i a l i s o l a t e d from the mycelium c o n t a i n e d much l e s s p r o t e a s e a c t i v i t y , and was a l s o e n r i c h e d i n F - I I component ( F i g u r e 1) r e l a t i v e t o s u p e r n a t a n t . A l s o chroma tography on h y d r o x y l a p a t i t e as d e s c r i b e d by Montgomery et al. ( 4 ) i s e f f e c t i v e i n removing c a r b o h y d r a t e and p r o t e a s e from l a r g o m y c i n F-II. NCI-FCRF P r o c e s s I I - A (Scheme 3). I n t h i s p r o c e s s the s t a r t i n g m a t e r i a l f o r the i s o l a t i o n of l a r g o m y c i n F - I I was the mycelium ( c e l l - p a s t e ) . The c e l l - p a s t e was suspended i n p o t a s s i u m phosphate b u f f e r , and the s u s p e n s i o n was s u b j e c t e d t o c e l l r u p t u r e by f r e e z e thaw s t e p s and r e - c e n t r i f u g e d o r f i l t e r e d . The crude l a r g o m y c i n complex was d i a l y s e d and c o n c e n t r a t e d and the c o n c e n t r a t e l o a d e d on t o a Sephadex G-100 column. The column was e l u t e d w i t h phosphate b u f f e r . Largomycin F - I I r i c h f r a c t i o n s were combined, d i a l y z e d , c o n c e n t r a t e d and l y o p h i l i z e d t o a l i g h t y e l l o w powder. The l a s t t r a c e s of c a r b o h y d r a t e i m p u r i t i e s were removed by a h y d r o x y l a p a t i t e column. Based on b i o l o g i c a l assay and HPLC p r o f i l e l a r g o m y c i n F - I I r i c h f r a c t i o n s were combined, d i a l y z e d , c o n c e n t r a t e d and l y o p h i l i z e d t o pure l a r g o m y c i n F - I I . R e a l i z i n g the d i f f i c u l t y of f r e e z i n g and thawing of l a r g e q u a n t i t i e s and the time t a k e n i n these o p e r a t i o n s another method was developed f o r the e x t r a c t i o n o f l a r g o m y c i n F - I I from mycelium. T h i s i s summarized i n Scheme 4. I t was found t h a t s o n i c a t i o n o r v i g o r o u s s t i r r i n g of the m y c e l i a w i t h d e i o n i z e d water o r 0.01 M phosphate b u f f e r l i b e r a t e d most of the l a r g o m y c i n F - I I i n the f i r s t two e x t r a c t i o n s (Scheme 4 ) . NCI-FCRF P r o c e s s I I - B (Scheme 5 ) . I n t h i s p r o c e s s the f r e e z e - t h a w s t e p of m y c e l i a s u s p e n s i o n , t o l i b e r a t e l a r g o m y c i n F - I I , of NCI-FCRF P r o c e s s I I - A has been r e p l a c e d by v i g o r o u s l y m i x i n g the m y c e l i a s u s p e n s i o n f o r 1 hour. The r e s u l t s of a 240 g a l l o n f e r m e n t a t i o n run are summarized i n Scheme 5. A comparison of the p r o p e r t i e s of t h e i s o l a t e d product w i t h the s t a n d a r d compound i s shown i n T a b l e I I , and t h e i r p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p a t t e r n i s shown i n F i g u r e 5. One problem encountered d u r i n g l a r g e s c a l e p u r i f i c a t i o n of l a r g o m y c i n F - I I was the c o n s t a n t p r e c i p i t a t i o n of aggregated mater i a l s , mainly i m p u r i t i e s , during u l t r a f i l t r a t i o n . This m a t e r i a l c o u l d not be removed by c e n t r i f u g a t i o n , o r f i l t r a t i o n through c e l i t e . M i c r o p o r o u s dead-ended f i l t e r s q u i c k l y became c l o g g e d . T h i s h i g h m o l e c u l a r weight m a t e r i a l c o u l d be e f f e c t i v e l y removed from the m a j o r i t y of the F - I I by passage of the F - I I through a 0.45 μ durapore membrane w i t h e x c e l l e n t r e c o v e r y and e f f i c i e n t removal of h i g h m o l e c u l a r weight p r o t e i n s . These r e s u l t s are summarized i n Scheme 6 and F i g u r e 6. The o t h e r a s p e c t of l a r g o m y c i n F - I I p u r i f i c a t i o n t h a t was r o u t i n e l y d i s c u s s e d w i t h i n the F e r m e n t a t i o n Program was whether i t was b e t t e r t o i s o l a t e F - I I from the s u p e r n a t a n t or from the myce-


144


Largomycin Whole Broth I Centrifuge

VII

Cell-Free Broth (Discard) Cell Paste

0

4


8

12

8

12

Min

Suspend 1 kg/liter in 0.01 M phosphate buffer, pH 7.0 Cell Suspension Freeze—Thaw (2 x) Centrifuge Cell Debris (Discard)

Crude Largomycin Solution 1 Dialyze/Concentrate

Dialysate/Concentrate out (Discard)

Concentrate

0

4 Min

Sephadex G-100 Elute with 0.01 M phosphate buffer, pH 7.0 Largomycin F-ll —Rich Fractions I Dialyze/Concentrate

LIL

Dialysate/Concentrate out (Discard) Lyophilize Largomycin F-ll Powder Hydroxylapatite Elute with 0.01 M—0.6 M gradient phosphate buffer, pM 7.0

I

^Dialyze/Concentrate F-ll Fractions Dialysate/Concentrate out (Discard)

Lyophilize

I Pure Largomycin F-ll |

0

4 Min

8

Scheme 3. E x t r a c t i o n and p u r i f i c a t i o n o f l a r g o m y c i n F - I I from c e l l - p a s t e (NCI-FCRF P r o c e s s I I - A ) . HPLC p a t t e r n o f the l a r g o mycin p u r i t y i s shown on the s i d e .



Scheme 4. G e n e r a l scheme f o r the e x t r a c t i o n of l a r g o m y c i n complex from the mycelium. Shaded peak r e p r e s e n t s the amount of l a r g o mycin F - I I i n the complex.



146

Largomycin Whole Broth (LM63, 240 gal)


Cell-Free Broth, 220 gal (Discard)

Cell Debris (Discard)

F-II

1 Centrifuge Cell Paste (-53 lbs) -60 g Suspend in 52 gal 0.01 M phosphate buffer Mix 1h (pH 7.0) Centrifuge

Crude Largomycin Solution (-47 gal) Dialyze/Concentrate Using Amicon DC-50

Dialysate/Concentrate out (Discard)

Concentrate (5.5L)

-54 g

Sephadex G-100 stack column Elute with 0.01 M phosphate buffer (140 L, pH 7.0) Largomycin F-II Rich Fractions (-11L) ι Dialyze/Concentrate 1 (Amicon DC-4) Dialysate/Concentrate out (Discard)

410 ml

- 10.6g

Hydroxylapatite column Elute with 0.01 M— 0.6M gradiant phosphate buffer * (PH 7.0) Pure Largomycin F-II Fractions (1.6L)

f Dialysate/Concentrate out (Discard)

Dialyze/Concentrate 750 ml

Lyophilize Pure Largomycin F-II (5.2g)

-5.2g

(Lot # LM63HA9-22-7) Scheme 5. Large s c a l e p u r i f i c a t i o n o f l a r g o m y c i n F - I I from p a s t e u s i n g modern methods (NCI-FCRF P r o c e s s I I - B ) .


cell-

PANDE Y ET AL.


Properties


• Appearance

• Solubility (0.1 mM Phosphate buffer) pH 7.0

Frederick (Lot # LM63HA9227)

Standard (RM-LM-F-II)

Flaky Yellow Solid

Granular Yellow Solid

Soluble

• Protein (%)

58

• Carbohydrate (%)

17.4

• MIC: M. luteus 0*g/ml) BIA (E. Coli strain 339)

30

• Protease (nmol/mg)

Partially Insoluble

44 9.5 100

0.6

5.0

0.33

0.33

• HPLC (1 mg/ml sol.)

T a b l e I I . Comparison of P h y s i c o c h e m i c a l P r o p e r t i e s of I s o l a t e d Largomycin F - I I

American Chemical Society Library 1155 16th st N. w. Weshtoflton, 0. Products; C. 20038 In Purification of Fermentation LeRoith, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.


148


F i g u r e 5. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p a t t e r n of 1 and 6, markers; 2, crude largomycin from c e l l l y s a t e ; 3, p u r i f i e d l a r g o mycin F - I I from f i l t e r e d b r o t h ; 4, Sephadex G-100 p u r i f i e d l a r g o mycin F - I I from c e l l l y s a t e ; 5, h y d r o x y l a p a t i t e column p u r i f i e d largomycin F - I I ( f i n a l product from c e l l l y s a t e ) .


9.

PANDEY ET AL.

LM 85-FD

149


(600 g, 6%-7%

F-ll)

+ 4 liters H 0


2

LM 85-30'

(11.2 mg/ml, ca. 4 liters, 44.8 g F-ll)

F-ll Permeates

Durapore LM 85-31 (8.1 mg/ml, ca. 3.3 liters = 26.7 g) + 2 liters H 0 2

Durapore LM 85-33

(5.2 mg/ml, ca. 2 liters = 10.5 g)

LM 85-35

(2.95 mg/ml, ca. 2 liters 5.9 g) 43.1 g

+ 2 liters H 0 2

Durapore • + 2 liters H 0 2

LM 85-36

(2 mg/ml, ca. 2.7 liters, 5.4 g F-ll)

Scheme 6.

Largomycin F - I I Durapore p r o c e s s i n g

scheme.




F i g u r e 6. High performance l i q u i d chromatography p r o f i l e of Durapore p r o c e s s i n g ( c o n d i t i o n s i m i l a r t o , as i n F i g u r e 1 ) .



9.

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151

lium. Crude m a t e r i a l i s o l a t e d from the mycelium was r e l a t i v e l y low i n p r o t e a s e s and i n h i g h m o l e c u l a r weight i m p u r i t i e s . Super n a t a n t p r o v i d e d much more raw m a t e r i a l i n i t i a l l y (up t o 300 yg/ml by HPLC) but t h i s F - I I was h i g h i n p r o t e a s e a c t i v i t y and was con taminated w i t h huge amounts of p r o t e i n a c e o u s m a t e r i a l from the medium. The e x p e r i e n c e s l e a r n e d i n the development of p r o c e s s I I , namely the a b i l i t y of h y d r o x y l a p a t i t e t o remove v e r y s i m i l a r impur i t i e s and the success of durapore membranes t o e f f e c t i v e l y remove l a r g e amounts of h i g h m o l e c u l a r weight c o n t a m i n a n t s , s p u r r e d f u r t h e r work on a s u p e r n a t a n t p r o c e s s . F e r m e n t a t i o n development work was a l s o p r o g r e s s i n g a t t h i s t i m e , and e f f e c t i v e s u b s t i t u t e s were found f o r the h i g h m o l e c u l a r weight components of the f e r m e n t a t i o n medium. S u b s t i t u t i o n of low m o l e c u l a r weight n i t r o g e n s o u r c e s s u b s t a n t i a l l y reduced the amount of h i g h m o l e c u l a r weight p r o t e i n i m p u r i t i e s p r e s e n t i n the s u p e r natant. A l s o about t h i s t i m e , we l e a r n e d t h a t l a r g o m y c i n F - I I s o l u t i o n s c o u l d be r a p i d l y c o n c e n t r a t e d u s i n g a wiped f i l m e v a p o r a t o r (WFE). Reduced p r e s s u r e low temperature e v a p o r a t i o n i s now r o u t i n e l y used f o r the c o n c e n t r a t i o n of l a r g e volumes (500 g a l . ) of crude l a r g o mycin F - I I s o l u t i o n s t o a more manageable volume (50 g a l . ) p r i o r t o ammonium s u l f a t e p r e c i p i t a t i o n or d i a f i l t r a t i o n procedures w i t h o u t the l o s s of b i o l o g i c a l a c t i v i t y . Parameters can be a d j u s t e d s o t h a t the m a t e r i a l b e i n g c o n c e n t r a t e d i s i n c o n t a c t w i t h the heated evapo r a t o r r o t a r chamber f o r no more than a few seconds. The product f e e d and the r e s u l t i n g c o n c e n t r a t e emerging from the e v a p o r a t o r a r e cooled. NCI-FCRF P r o c e s s I I I (Scheme 7 ) . T h i s i s e s s e n t i a l l y m o d i f i e d NCI-FCRF P r o c e s s I , where advantage of a l l our e x p e r i e n c e and the equipment a t hand was t a k e n t o i s o l a t e h i g h q u a l i t y l a r g o m y c i n F - I I from f i l t e r e d b r o t h . The f e r m e n t a t i o n b r o t h i s c l a r i f i e d by f i l t r a t i o n through c e l i t e i n a p l a t e and frame f i l t e r p r e s s , c o n c e n t r a t e d ca. 5 - f o l d w i t h a WFE and c e n t r i f u g e d t o remove s o l i d s . T h i s r i c h c o n c e n t r a t e i s d i a l y z e d and c o n c e n t r a t e d u s i n g 10 Κ h o l l o w f i b e r s on an Amicon DC-50. T h i s d i a l y z e d c o n c e n t r a t e i s t r e a t e d w i t h an e q u a l volume of s a t u r a t e d ammonium s u l f a t e , f i l t e r e d , and p a r t i a l l y d i a l y z e d and c o n c e n t r a t e d b e f o r e passage through Durapore c a s s e t t e s . The durapore permeate was c o n c e n t r a t e d and passed through a column of Sephadex G-100. Largomycin F - I I r i c h f r a c t i o n s a r e pooled c o n c e n t r a t e d and l y o p h i l i z e d o r immediately a p p l i e d t o a column of h y d r o x y l a p a t i t e w h i c h was e l u t e d w i t h 0.01 M phosphate b u f f e r . Largomycin F - I I r i c h f r a c t i o n s from t h i s f l a s h HA column were p o o l e d , c o n c e n t r a t e d by u l t r a f i l t r a t i o n and c a r e f u l l y chromato graphed on h y d r o x y l a p a t i t e . Pure l a r g o m y c i n f r a c t i o n s from t h i s column were p o o l e d , f i l t e r e d , and l y o p h i l i z e d t o g i v e a l i g h t y e l l o w , h i g h l y h y g r o s c o p i c powder t h a t was e q u i v a l e n t i n b i o a c t i v i t y and p u r i t y t o the cake d e r i v e d m a t e r i a l . T h i s f i r s t attempt w i t h s u p e r n a t a n t p r o c e s s produced ca. 15 g of h i g h q u a l i t y F - I I from about 400 g a l . of f e r m e n t a t i o n b r o t h . The procedure i s d e p i c t e d i n Scheme 7.



LM Fementation Broth Cake

Filter press Filtered Supernatant


0.45 μ Filter Cone. With Pellicon Door Oliver DC-50 DDS

Concentrate on WFE, approximately 5-fold WFE Concentrate Centrifuge, M-16s Rich Concentrate Dialyze/concentrate on DC-50 Dialyzed Concentrate Treat with equal volume sat. (NH ) S0 Mix, centrifuge M-16s 4

2

4

Clarified Concentrate Dialyze/concentrate on DC-50 Dialyzed Concentrate Pass through 0.45 μ Durapore filter Durapore Permeate Concentrate with 10K membrane on Pellicon Stack Starting Material Pass through Sephadex G-100 Pool F-ll rich fractions Concentrate with 10K membrane on Pellicon Stack Column Product Chromatograph on HA, 10 mM Flash column Pool F-ll rich fractions Concentrate with 10K membrane on Pellicon Flash HA Column Product Chromatograph on HA 0.1 mM to 20 mM gradient Pool F-ll rich fractions, concentrate, lyophilize Final Product Largomycin F-ll

Scheme 7. Largomycin F - I I supernatant r e c o v e r y p r o c e s s (NCI-FCRF Process I I I ) .


9.

PANDEY ET A L .


153

Conclusions


P r o c e s s development work on the p u r i f i c a t i o n of l a r g o m y c i n F - I I has r e s u l t e d i n two p r o c e s s e s , one from c e l l - p a s t e (NCI-FCRF P r o c e s s I I - B ) and the o t h e r from c e l l f r e e b r o t h (NCI-FCRF P r o cess I I I ) . Both the p r o c e s s e s have advantages over the Yamaguchi et àl. p r o c e s s i n the number o f s t e p s , s i m p l i c i t y and y i e l d i n g a b e t t e r b i o l o g i c a l l y a c t i v e p r o d u c t . Over 30 g of GMP q u a l i t y , homogeneous (on SDS g e l s ) b i o a c t i v e m a t e r i a l has been produced from a p p r o x i m a t e l y s i x s u c c e s s f u l m y c e l i a l r e c o v e r y runs and two s u p e r n a t a n t f e r m e n t a t i o n s . A p p r o x i m a t e l y 15 grams of these were d e r i v e d from 2-200 g a l l o n s u p e r n a t a n t r u n s , w i t h an o v e r a l l r e c o v e r y o f between 5-10%. T h i s m a t e r i a l i s c u r r e n t l y undergoing f o r m u l a t i o n and t o x i c i t y s t u d i e s a t the N a t i o n a l Cancer I n s t i t u t e . Acknowledgments T h i s r e s e a r c h was sponsored by the P u b l i c H e a l t h S e r v i c e , N a t i o n a l Cancer I n s t i t u t e under C o n t r a c t No. NO-l-CO-75380 and NO-l-CO-23910. We thank D r s . John Douros and Matthew S u f f n e s s of the N a t i o n a l Cancer I n s t i t u t e and a l l the p e r s o n n e l o f F e r m e n t a t i o n Program, NCI-FCRF, who were i n v o l v e d w i t h t h i s p r o j e c t a t one time or the o t h e r f o r a l l t h e i r h e l p , i n t e r e s t and encouragement.

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Yamaguchi, T.; Furumai, T.; Sato, M.; Okuda, T; Ishida, N. J. Antibiot. 1970, 23, 369-372. 2. Yamaguchi, T.; Kashida, T.; Nawa, K.; Yajma, T.; Miyagishima, T.; Ito, Y.; Okuda, T.; Ishida, N.; Kumagai, K. J. Antibiot. 1970, 23, 373-381. 3. Yamaguchi, T.; Seto, M.; Oura, Y.; Arai, Y.; Enomoto, K.; Ishida, N.; Kumagai, K. J. Antibiot. 1970, 23, 382-387. 4. Vandre, D.D.; Zaheer, Α.; Squier, S.; Montgomery, R. Biochemistry 1982, 21, 5089-5096. 5. Elespuru, R.K.; Yarmolinsky, M.B. Environ. Mutagen. 1979, 1, 65-78. 6. Elespuru, R.K.; White, R.J. Cancer Res. 1983, 43, 2819-2830. 7. Ishida, N.; Miyazaki, K.; Kumagai, K.; Rikimaru, M. J. Antibiot. 1965, Ser. A. 18, 68-76. 8. Meienhofer, J.; Maeda, H.; Glaser, C.B.; Czombos, J.; Kuromizu, K. Science 1972, 178, 875-876. 9. Napier, M.A.; Holmquist, B.; Strydom, D.J.; Goldberg, I.H. Biochem. Biophys. Res. Commun. 1979, 89, 635-642. 10. Chimura, H.; Ishizuka, M.; Hamada, M.; Hori, S.; Kimura, K.; Iwanaga, J.; Takeuchi, T.; Umezawa, H. J. Antiobiot. 1968, 21, 44-49. 11. Yamashita, T.; Naoi, N.; Watanabe, K.; Takeuchi, T.; Umezawa, H. J. Antiobiot. 1976, 29, 415-423. 12. Naoi, N.; Miwa, T.; Okazaki, T.; Watanabe, K.; Takeuchi, T.; Umezawa, H. J. Antibiot. 1982, 35, 806-813. RECEIVED September 11, 1984


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