Process for the whole broth extraction of avermectin

Europaisches Pa ten tamt

European Patent Office © Publication number: 0 0 8 2 6 7 4

Office europeen des brevets A 2

© EUROPEAN PATENT APPLICATION

© Application number: 82306704.6 © Int. CI.3: C 07 H 17 /08

^ C 12 P 19 /62 @ Date of filing: 15.12.82

© Priority: 21.12.81 US 332418 @ Applicant: MERCK & CO. INC. 126, East Lincoln Avenue P.O. Box 2000 Rahway New Jersey 07065(US)

© Date of publication of application: 29.06.83 Bulletin 83/26 © Inventor: Bagner.Carl

144 Howiand Avenue © Designated Contracting States: Paramus New Jersey(US)

AT BE CH DE FR GB IT LI LU NL SE @ Inventor: Wildman, Arthurs.

33 Hillcrest Road Martinsville New Jersey 08836(US)

© Representative: Crampton, Keith John Allen et al, D YOUNG & C0 10 Staple Inn London WC1V7RDIGB)

© Process for the whole broth extraction of avermectin. © In the whole broth extraction of avermectins, the pH of the whole broth is adjusted with mineral acid and the broth is then contacted with a solvent extractant so that the avermec- tin-active component is taken up by the solvent. The new avermectin-rich extractant is then processed through a solvent concentration step and the avermectin isolated by conventional crystallization. The extractant is then recycled with appropriate make-up to extract a subsequent batch of whole broth.

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I n the whole broth extraction of avermectins, the pH of the whole broth is adjusted with mineral acid and the broth is then contacted with a solvent extractant so that the avermec- tin-active component is taken up by the solvent. The new avermectin-rich extractant is then processed through a solvent concentration step and the avermectin isolated by conventional crystallization. The extractant is then recycled with appropriate make-up to extract a subsequent batch of whole broth.

This invention relates to the ext rac t ion of whole broth of

avermect ins , which are products produced by known fermenta t ion methods ,

for example, see U.S. Patent No. 4,199,579. In the past, the whole b ro th

was filtered using a filter aid. The resulting wet filter cake was t h e n

contacted with an extract ing agent usually toluene, and then the water was

removed by azeotropic distillation. The slurry comprising the e x t r a c t a n t ,

the mycelia and the filter aid material was then refil tered and t h e

ex t rac tan t solution of avermect in concen t ra ted and crystall ized. The

major disadvantage of this prior process is that is requires the use of a f i l t e r

aid, which can not be disposed of until after the final separation step. This

presents serious problems of waste disposal because the avermectins, when

highly concent ra ted , are potentially toxic materials . This, in the past t h e

disposal of the filter aid, which c o n t a i n e d potentially toxic res idual

avermectin, const i tuted a disposal hazard that was solved only by

incincerat ion or other such drastic disposal means .

In accordance with the present invention, an avermec t in - r ich f r a c t i on

is separated from an avermec t in -con ta in ing whole f e rmenta t ion broth by

acidifying the whole broth to a pH of from 1.5 to 5; admixing the ac id i f ied

whole broth with an ex t r ac t an t in which avermect ins are soluble in a r a t i o

of ex t r ac t an t to broth of the least 0.2 by volume; heating the admixture of

whole broth and ex t r ac t an t to a t empera tu re of from about 20°C to 100°C

or to the reflux t empera tu re of the ex t rac tan t in a first stage for a period

of at least one hour; decanting the a v e r m e c t i n - c o n t a i n i n g e x t r a c t a n t

fract ion from the admixture; stripping the ex t rac tan t from the a v e r m e c t i n -

containing ex t r ac t an t fraction; and collecting the a v e r m e c t i n .

The present invention thus eliminates the need to filter the whole

broth and hence el iminates the use of any filter aid material since t h e

ex t rac t ion is performed directly upon an avermect in whole broth .

Fur the rmore , to avoid toxicity problems associated with their m a n u f a c t u r e ,

though not their use, the manufactur ing can here by conducted in an i sola ted

closed system and only cryst ra l l ized material removed from the sys tem

during the packaging step. The process of the invention also el iminates any

problems associated with the disposal of spent mycelia. The invention can

be t te r be understood by reference to the accompanying drawing which is a

flow chart depicting the process .

B r i e f l y , t he p r o c e s s of t h i s i n v e n t i o n

e n t a i l s a d j u s t i n g the pH of t h e w h o l e b r o t h w i t h i n

t h e r a n g e of 1 .5 to 6, more d e s i r a b l y w i t h i n t h e

r a n g e pH 2 to 4 . 2 and m o s t p r e f e r a b l y w i t h i n t h e pH

r a n g e 3 to 4 w i t h m i n e r a l a c i d . A pH in e x c e s s of 7

i s to be a v o i d e d s i n c e n e u t r a l or b a s i c c o n d i t i o n s

d e c o m p o s e t h e a v e r m e c t i n . The a c i d e m p l o y e d can b e

any m i n e r a l a c i d s u c h as s u l f u r i c , h y d r o c h l o r i c ,

n i t r i c , or o t h e r s t r o n g a c i d , a l t h o u g h s u l f u r i c i s

p r e f e r r e d . The w h o l e b r o t h is i n t i m a t e l y c o n t a c t e d

w i t h and a g i t a t e d w i t h an e x t r a c t a n t e x e m p l i f i e d b y

t o l u e n e and t h e a v e r m e c t i n . c o m p o n e n t s d i s s o l v e d i n t o

and t a k e n up by the e x t r a c t a n t . The s o l u t i o n

c o m p r i s i n g t he a v e r m e c t i n and the e x t r a c t a n t i s t h e n

p a s s e d t h r o u g h a s e p a r a t o r to s t r i p o f f t h e

e x t r a c t a n t , and t he r e m a i n i n g a v e r m e c t i n i s o l a t e d b y

c r y s t a l l i z a t i o n or o t h e r a p p r o p r i a t e means known i n

t h e a r t . The s t r i p p e d o f f e x t r a c t a n t is r e c y c l e d t o

p e r f o r m i t s f u n c t i o n on a s u b s e q u e n t b a t c h of w h o l e

b r o t h . The p r o c e s s w i l l now be d i s c u s s e d in d e t a i l .

I n t o an a p p r o p r i a t e v e s s e l t h e r e i s fed t h e

w h o l e b r o t h f rom an a v e r m e c t i n f e r m e n t a t i o n t h a t h a s

been a d m i x e d w i t h s u f f i c i e n t m i n e r a l a c i d to a d j u s t

t h e pH of t he w h o l e b r o t h to b e t w e e n 1 .5 and 2 t o

6 . 0 . A f t e r s u f f i c i e n t a g g i t a t i o n to f u l l y a d j u s t t h e

pH of t h i s w h o l e b r o t h , a d d i n g a d d i t i o n a l i n c r e m e n t s

of a c i d as n e c e s s a r y , t h e r e i s a d d e d to t he pH a d j u s t e d b r o t h s u f f i c i e n t e x t r a c t a n t such t h a t t h e

e x t r a c t a n t to b r o t h r a t i o i s f rom 0 .2 to 1 .0 o r

g r e a t e r in v o l u m e . A r a t i o of 2 or 3 can b e

s a t i s f a c t o r i l y u s e d , b u t w i l l g e n e r a l l y i n t r o d u c e

more e x t r a c t a n t t h a n n e c e s s a r y . I f d e s i r e d , a

c o m m e r c i a l l y a v a i l a b l e f l o c c u l e n t can t h e n be a d d e d

to a c h i e v e a more e f f i c i e n t e x t r a c t i o n of t he w h o l e

b r o t h .

The a d m i x t u r e of w h o l e b r o t h , e x t r a c t a n t a n d

o p t i o n a l f l o c c u l e n t is h e a t e d by a n a p p r o p r i a t e

m e a n s , p r e f e r a b l y a s t e a m j a c k e t to a t e m p e r a t u r e o f

f rom 20°C to 100°C or to t he r e f l u x t e m p e r a t u r e o f

the e x t r a c t a n t . U n l e s s a p p r o p r i a t e c o n t a i n m e n t a n d

p r e s s u r i z a t i o n means a r e e m p l o y e d , t h e t e m p e r a t u r e t o

w h i c h t h e a d m i x t u r e is h e a t e d s h o u l d n o t e x c e e d t h e

r e f l u x t e m p e r a t u r e of the e x t r a c t a n t e m p l o y e d . T h e

s e q u e n c e of a d d i t i o n of a c i d and e x t r a c t a n t is n o t

c r i t i c a l and i f d e s i r e d , t he e x t r a c t a n t can be a d d e d

a f t e r h e a t i n g t he a c i d i f i e d w h o l e b r o t h . A f t e r

e i t h e r c o n t i n u o u s l y or i n t e r m i t t e n t l y a g i t a t i n g t h e

h e a t e d w h o l e b r o t h - e x t r a c t a n t a d m i x t u r e f o r a b o u t 1 - 4

h o u r s or more i t i s a l l o w e d to s e t t l e . A f t e r

s e t t l i n g , t h e a v e r m e c t i n e n r i c h e d e x t r a c t a n t f r a c t i o n

is t h e n d e c a n t e d and t r a n s f e r r e d to an a p p r o p r i a t e

v e s s e l f o r c r y s t a l l i z a t i o n .

W h i l e t h i s d e c a n t a t i o n s t e p can be p e r f o r m e d in a s i n g l e v e s s e l , i t is g e n e r a l l y p r e f e r r e d t o

t r a n s f e r t he a d m i x t u r e to e q u i p m e n t more a p p r o p r i a t e to p e r f o r m a d e c a n t a t i o n . T h u s , t he d e c a n t e d

a v e r m e c t i n r i c h e x t r a c t a n t is m o s t p r e f e r a b l y

t r a n s f e r e d to a c o n c e n t r a t o r w h e r e b y means w e l l

known in t h e a r t t he e x t r a c t a n t i s s t r i p p e d f rom t h e

f r a c t i o n l e a v i n g an o i l c o n t a i n i n g t h e a v e r m e c t i n

c o m p o n e n t s . The r e c o v e r e d e x t r a c t a n t i s t h e n

r e t u r n e d to t h e b e g i n n i n g of t he p r o c e s s t o g e t h e r

w i t h wha t m a k e - u p may be r e q u i r e d to b r i n g t h e

e x t r a c t a n t b r o t h r a t i o to t he d e s i r e d l e v e l .

The n o n d e c a n t a b l e r e s i d u e i s t r e a t e d a s

f o l l o w e s , p r e f e r a b l y by b e i n g fed to a s e c o n d m i x e r

w h e r e i t a g a i n i s a g i t a t e d and h e a t e d g e n e r a l l y

w i t h i n the same t i m e p e r i o d and t e m p e r a t u r e s a s

p r e v i o u s l y d e s c r i b e d in r e g a r d to t he w h o l e b r o t h

e x t r a c t a n t a d m i x t u r e .

A f t e r h e a t i n g and a g i t a t i n g fo r t he d e s i r e d

p e r i o d , t he s p e n t b r o t h and e x t r a c t a n t a r e s e p a r a t e d

f rom the r e s i d u e . Most p r e f e r a b l y the r e s i d u e i s

t r a n s f e r r e d f r o m t h e s e c o n d m i x e r to a s e p a r a t o r

w h e r e the e x t r a c t a n t is d e c a n t e d and r e c y c l e d to t h e

i n i t i a l s t e p . T h i s d e c a n t a t i o n s t e p c a n , i f d e s i r e d ,

be p e r f o r m e d in t h e s e c o n d m i x e r , b u t i s p r e f e r a b l y

done in e q u i p m e n t e s p e c i a l l y s u i t e d to r e m o v a l of t h e

e x t r a c t a n t f r a c t i o n f rom t he r e s i d u e .

W h a t e v e r t h e n r e m a i n s f rom t he s e c o n d m i x e r

or a s u b s e q u e n t s e p a r a t i o n v e s s e l is s p e n t b r o t h

c o m b i n e d w i t h s m a l l q u a n t i t i e s of e x t r a c t a n t a n d

f l o c c u l e n t m a t e r i a l . T h e s e r e m a i n s a r e t h e n fed to a

s t r i p p e r w h e r e w h a t e v e r r e m a i n i n g e x t r a c t a n t can b e

r e c o v e r e d . T h i s i s s u i t a b l y a c c o m p l i s h e d by h e a t i n g

t he r e m a i n s a b o v e the r e f l u x t e m p e r a t u r e of t h e

e x t r a c t a n t . W h a t e v e r e x t r a c t a n t t h a t comes o f f i s

r e t u r n e d to t h e f i r s t or s e c o n d m i x e r , a n d

p r e f e r a b l y t h e s e c o n d .

W h a t e v e r r e s i d u e e m i n a t e s f rom the s t r i p p e r i s

f ed to a c a u s t i c b a t h h e a t e d to a t e m p e r a t u r e of f r o m

80° to 1 0 0 ° C . T h e r e i n t he s p e n t b r o t h i s s u b j e c t e d

to f rom 1/2 to 2 h o u r s of h e a t i n g a t a pH 8 a n d

p r e f e r a b l y a b o u t a pH of 12. T h i s e x p o s u r e to h e a t

a t e l e v a t e d pH d e c o m p o s e s t he a v e r m e c t i n s and r e n d e r s

the d i s c h a r g e of t h e s p e n t b r o t h n o n - h a z a r d o u s .

The f o l l o w i n g is a d e s c r i p t i o n of t h e

p r e f e r r e d mode of c a r r y i n g o u t t h i s i n v e n t i o n .

A b a t c h of a v e r m e c t i n w h o l e b r o t h c o n t a i n i n g

a b o u t 40 Kg. ( p u r e b a s i s ) of a v e r m e c t i n was e x t r a c t e d

by t he f o l l o w i n g p r o c e d u r e : The b r o t h was a d j u s t e d

to pH 2 .5 w i t h d i l u t e H2S04 in t he h a r v e s t t a n k

and t r a s f e r r e d to a 2 0 , 0 0 0 g a l l o n e x t r a c t o r w h e r e i t

was h e a t e d to 80°C . A s e c o n d pH a d j u s t m e n t may b e

r e q u i r e d . A f t e r a 2 hou r h e a t t r e a t m e n t

a p p r o x i m a t e l y 4000 (20% of b r o t h v o l u m e ) g a l l o n s o f

l e a n t o l u e n e f rom t h e p r e v i o u s b a t c h was a d d e d . T h e

b a t c h was e x t r a c t e d f o r 3 h o u r s . J u s t p r i o r to t h e

end of t he e x t r a c t i o n p e r i o d t h e pH is r e a d j u s t e d t o

5 .5 w i t h a q u e o u s NaOH. The c a u s t i c a d d i t i o n was m a d e

u s i n g an e d u c t o r so t h a t t he 50% c a u s t i c u s e d i s

d i l u t e d w i t h w a t e r b e f o r e r e a c h i n g t he b a t c h . S i x

g a l l o n s of f l o c c u l a t i n g a g e n t ( C a l g o n WT-2640 w a s t h e n a d d e d . L e s s e r or g r e a t e r a m o u n t s o f

f l o c c u l a t i n g a g e n t can be e m p l o y e d . The b a t c h w a s t h e n f ed t h r o u g h a two s t a g e c o n t i n u o u s e x t r a c t i o n

t r a i n . R i c h t o l u e n e and e x t r a c t e d w h o l e b r o t h w e r e

s e p a r a t e d in a d e c a n t e r . The r i c h t o l u e n e c o n t a i n i n g a b o u t 95% of t h e b a t c h e s a v e r m e c t i n c o n t e n t was s e n t

f o r i s o l a t i o n of p r o d u c t . E x t r a c t e d b r o t h was t h e n

m i x e d w i t h t o l u e n e in a s e c o n d s t a g e m i x e r . T h i s

m i x t u r e o v e r f l o w e d to a d e c a n t e r w h e r e l e a n t o l u e n e

and s p e n t b r o t h were s e p a r a t e d . Lean t o l u e n e w a s

t h e n c o l l e c t e d fo r u se in t he n e x t b a t c h .

S u i t a b l e e x t r a c t a n t s f o r use in t h i s

i n v e n t i o n i n c l u d e t h o s e o r g a n i c s o l v e n t s t h a t a r e n o t

m i s c i b l e w i t h w a t e r to any a p p r e c i a b l e d e g r e e and a r e

s o l v e n t s f o r and i n e r t to r e a c t i o n w i t h a v e r m e c t i n o r

o t h e r c o m p o n e n t s of t h e e x t r a c t i o n p r o c e s s . T h e s e

i n c l u d e t o l u e n e , x y l e n e , and o t h e r a r o m a t i c s

i n c l u d i n g b e n z e n e , a l t h o u g h b e n z e n e is g e n e r a l l y t o

be a v o i d e d b e c a u s e of i t s s u s p e c t e d c a r c i n o g e n i c i t y .

O t h e r s u i t a b l e s o l v e n t s i n c l u d e the h a l o g e n a t e d

a r o m a t i c s such as c h l o r o t o l u e n e , c h l o r o b e n z e n e , a n d

t h e l i k e , as w e l l as o t h e r n o n - a r o m a t i c s o l v e n t s s u c h

as N - b u t a n o l and o t h e r a l c o h o l s of f rom 4-8 c a r b o n

a t o m s as w e l l as m e t h y l e n e c h l o r i d e .

Now h a v i n g d e s c r i b e d the i n v e n t i o n , t h e r e

w i l l become e v i d e n t v a r i e n t s w h i c h a r e o b v i o u s to o n e s k i l l e d in the a r t , w h i c h v a r i e n t s do n o t d e p a r t f r o m

the s p i r i t of t he f o l l o w i n g c l a i m s .

1 A process for separating an ave rmec t in - r i ch fraction from an

avermec t in -con ta in ing whole fermenta t ion broth comprising acidifying t h e

whole broth to a pH of from 1.5 to 5; admixing the acidified whole b ro th

with an ex t r ac tan t in which avermect ins are soluble in a ratio of e x t r a c t a n t

to broth of the least 0.2 by volume; heating the admixture of whole b ro th

and ex t r ac tan t to a t empera tu re of from about 20°C to 100°C or to t h e

reflux t empera tu re of the ex t rac tan t in a first stage for a period of at l e a s t

one hour; decanting the avermect in-conta in ing ex t r ac t an t fraction from t h e

admixture; stripping the ex t rac tan t from the avermect in - con ta in ing

ex t r ac t an t fraction; and collecting the a v e r m e c t i n .

2 A process according to claim 1 where the ex t r ac t an t is to luene .

3 A process according to claim 1 or 2 where the ex t r ac t an t to b ro th

ratio is from 0.2:1 to 3:1 by volume.

4 A process according to claim 3 where the ratio is from 0.2:1 to 1:1.

5 A process according to any preceding claim where the heating is

accompanied by ag i t a t ion .

6 A process according to any preceding claim where the heating is

conducted for a period of from 1 to 4 hours.

7 A process according to any preceding claim where the pH is in t h e

range 2 to 4.2.

8 A process according to any preceding claim where the s t r ipped

ex t rac tan t is recycled to a first stage to ext rac t a subsequent a l iquot

portion of whole b ro th .

9 A process according to any preceding claim where the residue f r o m

the decantat ion step is heated in a second stage to a t empera tu re up to t h e

reflux t empera tu re of the ex t rac tan t or 100 C for a period of at least 1

hour and then the ex t rac tan t fraction is decanted of f .

1 0 A process according to claim 9 where the decanted ex t rac tan t is

recycled to extract a subsequent aliquot portion of whole broth .