Continuous Purification of Proteins by Selective Nonadsorptive

Chapter 2

Continuous Purification of Proteins by Selective Nonadsorptive Preparative Chromatography

Downloaded by STANFORD UNIV GREEN LIBR on August 2, 2012 | http://pubs.acs.org Publication Date: May 24, 1993 | doi: 10.1021/bk-1993-0529.ch002

T. K. Nadler and F. E. Regnier Department of Chemistry, School of Science, Purdue University, West Lafayette, IN 47907

Continuous forms of chromatography are important to the biotechnology industry for the production of therapeutic proteins. Selective non-adsorption preparative (SNAP) chromatography may be used in a cross-current, simulated moving bed to produce a continuous purification system. It has the advantages of speed, efficient use of packing material, scalability, decrease cost of pumping systems, gentleness with regard to protein denaturation, and reduced dilution of the sample. An immobilized metal affinity chromatography (IMAC) column may be added in tandem with SNAP to concentrate as well as purify the protein. T h e r e i s a g r e a t need i n t h e b i o t e c h n o l o g y i n d u s t r y f o r s e p a r a t i o n t e c h n o l o g y o f i n c r e a s e d f l e x i b i l i t y , s p e c i f i c t h r o u g h p u t , and economic e f f i c i e n c y t h a t may be used i n t h e p r o d u c t i o n o f t h e r a p e u t i c p r o t e i n s . These a t t r i b u t e s a r e o f t e n a s s o c i a t e d w i t h c o n t i n u o u s p r o c e s s e s and i s t h e r e a s o n t h a t c o n t i n u o u s p u r i f i c a t i o n systems a r e now o f g r e a t interest. Methods f o r continuous purification o f p r o t e i n s by chromatographic, e l e c t r o p h o r e t i c , aqueous two-phase e x t r a c t i o n and reversed m i c e l l e separation technology are c u r r e n t l y being explored. T h i s paper f o c u s e s b r i e f l y on c o n t i n u o u s c h r o m a t o g r a p h i c t e c h n i q u e s i n g e n e r a l and p r i m a r i l y on a new c o n t i n u o u s c h r o m a t o g r a p h i c technique c a l l e d s e l e c t i v e n o n - a d s o r p t i o n p r e p a r a t i v e chromatography (SNAP).

Continuous Forms of Chromatography C o n t i n u o u s c h r o m a t o g r a p h i c s e p a r a t i o n s were f i r s t w i d e l y used i n t h e p e t r o l e u m i n d u s t r y (1,2) where work i n i t i a l l y c o n c e n t r a t e d on t h e development o f l a r g e - s c a l e b a t c h s e p a r a t i o n s . Continuous systems became f a v o r e d because t h e y were more flexible, c o u l d be r u n u n a t t e n d e d , d e c r e a s e t h e need f o r r e c y c l i n g e f f l u e n t , and u t i l i z e d s o r b e n t m a t e r i a l s more e f f e c t i v e l y . Much o f t h i s work has been a p p l i e d r e c e n t l y i n t h e development o f c o n t i n u o u s c h r o m a t o g r a p h i c methods f o r proteins (3-5).

0097-6156/93/0529-0014$06.00/0 © 1993 American Chemical Society

In Chromatography in Biotechnology; Horváth, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Continuous Purification of Proteins

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M u l t i p l e approaches have been t a k e n i n t h e c o n s t r u c t i o n o f c o n t i n u o u s chromatography systems. Counter-current flow, c r o s s c u r r e n t f l o w , c o - c u r r e n t f l o w , c o n t i n u o u s s t i r r e d t a n k s , and s e l e c t i v e n o n - a d s o r p t i o n a r e a l l examples o f c o n t i n u o u s systems. Counter-Current Flow Separations. These systems a r e based on t h e m o b i l e phase and s o r b e n t moving i n o p p o s i t e d i r e c t i o n s as t h e name i m p l i e s . Because c o u n t e r - c u r r e n t systems g e n e r a l l y s e p a r a t e a m i x t u r e i n t o o n l y two f r a c t i o n s , s e l e c t i v i t y i s v e r y i m p o r t a n t w i t h complex mixtures. Moving beds i n which b o t h t h e s o r b e n t and s o l v e n t a r e t r a n s p o r t e d (1,2), s i m u l a t e d moving beds i n which t h e s o r b e n t i n a f i x e d bed appears t o move t h r o u g h t h e use o f v a l v e s a l o n g t h e column a x i s (6,7), a moving column approach t h a t u s e s column s w i t c h i n g t o s i m u l a t e s o r b e n t t r a n s p o r t (8,9), and f l u i d i z e d bed chromatography (10,11) a r e a l l forms o f c o u n t e r - c u r r e n t s e p a r a t i o n s . The p r o b l e m w i t h these systems r e l a t i v e t o p r o t e i n s i s t h a t t h e y a r e g e n e r a l l y i s o c r a t i c e l u t i o n systems and p r o t e i n s a r e not e a s i l y s e p a r a t e d i s o c r a t i c a l l y . Cross-Current Flow Separations. A n n u l a r chromatography i s t h e most t y p i c a l example o f a c r o s s - c u r r e n t f l o w system (12-16). Rotating a n n u l a r columns, i n which t h e s o r b e n t i s p l a c e d between two c o n c e n t r i c c y l i n d e r s t o form an a n n u l a r column, have been e f f e c t i v e i n s i z e e x c l u s i o n s e p a r a t i o n s o f p r o t e i n s ( 5 ) . Moving column systems w i t h a r o t a t i n g b u n d l e o f columns a r e a n o t h e r v e r s i o n o f t h i s approach (13,17,18). L i q u i d i n t h e moving column system i s d e l i v e r e d t o t h e columns and e l u e n t t o t h e c o l l e c t i o n f l a s k s by l i q u i d d i s t r i b u t o r s . A t the p r e s e n t t i m e o n l y i s o c r a t i c s e p a r a t i o n s o f p r o t e i n s have been a c h i e v e d w i t h t h e s e systems. I t does not appear t h a t t h e s p e c i f i c t h r o u g h p u t o f t h e s e systems i s any h i g h e r t h a n t h a t o f t h e i n d i v i d u a l columns c o m p r i s i n g t h e system. Moving Belt Separation. A v e r y unique system has been c o n s t r u c t e d by u s i n g a moving mylar b e l t on which n y l o n pouches o f immunosorbent were a t t a c h e d (19). The pouches resembled t e a bags o f immunosorbent t h r o u g h which p r o t e i n s c o u l d d i f f u s e w i t h o u t l o o s i n g t h e a d s o r b e n t . The b e l t was f i r s t d i p p e d i n t o t h e a s s o c i a t i o n chamber where i t s e l e c t i v e l y bound t h e a n t i g e n o f i n t e r e s t ( i n t h i s c a s e a l k a l i n e p h o s p h a t a s e ) . As t h e b e l t e x i t e d each chamber, t h e pouches were squeezed by a r o l l e r t o remove e x c e s s liquid. Unbound p r o t e i n was removed and antigen c o l l e c t e d by s e q u e n t i a l p a s s a g e t h r o u g h a wash chamber and d i s s o c i a t i o n chamber r e s p e c t i v e l y . Subsequent passage t h r o u g h a n o t h e r wash chamber r e c y c l e d t h e s o r b e n t pouches. A system u s i n g 5 g o f immunosorbent was c a p a b l e o f a t l e a s t a 10 f o l d p u r i f i c a t i o n w i t h 90% r e c o v e r y and a 35-40 h r c y c l e t i m e (19). Although continuous, the s p e c i f i c throughput o f t h i s system was v e r y low. I t i s q u e s t i o n a b l e whether t h i s system i s e a s i l y s c a l e d up. Continuous S t i r r e d Tank Reactors (CSTR). These systems have been used in s i t u a t i o n s where one o r two equilibrium contact stages are s u f f i c i e n t f o r a h i g h y i e l d s e p a r a t i o n (3,20,21). Each stage, or c o n t a c t o r , i s e q u i v a l e n t t o one t h e o r e t i c a l p l a t e . The CSTR must have a t l e a s t two s t a g e s , one f o r a d s o r p t i o n and a n o t h e r f o r d e s o r p t i o n o r r e c y c l i n g t o be c o n t i n u o u s . A d s o r b e n t must be s e p a r a t e d from t h e s o l v e n t i n each s t a g e . I t i s t h i s s e p a r a t i o n p r o c e s s where CSTR systems d i v e r g e i n d e s i g n . The c o n t i n u o u s a f f i n i t y - r e c y c l e e x t r a c t i o n


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CHROMATOGRAPHY IN BIOTECHNOLOGY

(CARE) system o f Gordon (3) i s a good example o f a CSTR p u r i f i c a t i o n system. 6 - g a l a c t o s i d a s e from Ε, Coli was p u r i f i e d 18 f o l d w i t h 79% r e c o v e r y i n a two s t a g e CARE system u s i n g an a f f i n i t y s o r b e n t f o r 6 - g a l a c t o s i d a s e (PABTG/Agarose). A d d i t i o n o f a wash s t a g e between t h e a d s o r b i n g and d e s o r b i n g s t a g e i n c r e a s e d t h e p u r i f i c a t i o n t o 22 f o l d and recovery t o 90%. In a c o u n t e r - c u r r e n t a d s o r p t i o n d e s i g n , the purification jumped t o 170 f o l d , but r e c o v e r y fell t o 72% (3). O p t i m i z a t i o n o f t h e s e systems i s c r i t i c a l . D r a s t i c changes i n y i e l d and r e c o v e r y may be o b t a i n e d w i t h minor a l t e r a t i o n s o f system d e s i g n and o p e r a t i o n .


S e l e c t i v e N o n - A d s o r p t i o n P r e p a r a t i v e (SNAP) Chromatography C h r o m a t o g r a p h i c s e p a r a t i o n s o f p r o t e i n s a r e a c h i e v e d by d i f f e r e n t i a l adsorption at surfaces in all cases except size-exclusion chromatography. These s u r f a c e m e d i a t e d s e p a r a t i o n s a r e g e n e r a l l y b a s e d on s e l e c t i v e e l u t i o n o f s u b s t a n c e s from a s o r b e n t s u r f a c e . E l u t i o n i s a c h i e v e d i n e i t h e r an i s o c r a t i c o r g r a d i e n t e l u t i o n r e g i m e . Because p r o t e i n s adsorb t o s u r f a c e s a t m u l t i p l e s i t e s , v e r y s m a l l changes i n s o l v e n t c o m p o s i t i o n have a l a r g e impact on t h e a d s o r p t i o n / d e s o r p t i o n e q u i l i b r i u m and i s o c r a t i c e l u t i o n i s seldom used . E l u t i o n i s more commonly a c h i e v e d w i t h a s o l v e n t g r a d i e n t o f i n c r e a s i n g d e s o r b i n g strength. U n f o r t u n a t e l y g r a d i e n t e l u t i o n i s a problem i n continuous chromatography t h a t would be d e s i r a b l e t o circumvent. Protein d e n a t u r a t i o n i s a n o t h e r problem i n c h r o m a t o g r a p h i c systems. Some p r o t e i n s d e n a t u r e when t h e y a r e adsorbed t o a s o r b e n t s u r f a c e ( 2 2 ) . I t would be d e s i r a b l e t o p u r i f y p r o t e i n s w i t h o u t h a v i n g t o adsorb them t o t h e chromatography column. The f a c t t h a t p r o t e i n s a r e p u r i f i e d by a s e r i e s of d i s c r e t e p u r i f i c a t i o n steps i s yet another negative f e a t u r e of c u r r e n t s e p a r a t i o n systems. M u l t i p l e step p u r i f i c a t i o n s are both l a b o r i n t e n s i v e and d i m i n i s h r e c o v e r y . I t would be d e s i r a b l e t o combine s t e p s i n p r o t e i n p u r i f i c a t i o n . The s e l e c t i v e n o n - a d s o r p t i o n p r e p a r a t i v e chromatography (SNAP) approach d e s c r i b e d below i s an e f f o r t t o a d d r e s s t h e s e problems i n t h e c a s e o f p r e p a r a t i v e chromatography. SNAP chromatography i s u n i q u e l y d i f f e r e n t from o t h e r forms o f chromatography i n t h a t i t removes a l l o f t h e p r o t e i n s i n a sample e x c e p t t h e one o f i n t e r e s t . The t a r g e t p r o t e i n i s a l l o w e d t o p a s s through the column u n r e t a i n e d . The ion-exchange form o f SNAP chromatography was first reported by Petrilli et a l . for the p u r i f i c a t i o n o f a s p a r t a t e a m i n o - t r a n s f e r a s e ( 2 3 ) . The system s e p a r a t e s p r o t e i n s by t a k i n g advantage o f t h e o b s e r v a t i o n t h a t r e t e n t i o n o f a p r o t e i n on ion-exchange chromatography columns i s m i n i m a l when i t i s a t i t s i s o e l e c t r i c p o i n t ( p i ) . ( I t w i l l be r e c a l l e d t h a t t h e n e t c h a r g e of a p r o t e i n i s z e r o a t i t s p i . ) At pH v a l u e s above i t s p i , a p r o t e i n has a net n e g a t i v e c h a r g e and w i l l adsorb t o an a n i o n exchange s o r b e n t (Figure 1). In c o n t r a s t , t h e p r o t e i n i s p o s i t i v e l y c h a r g e d and w i l l a d s o r b t o a c a t i o n e x c h a n g i n g m a t r i x below i t s p i . Retention of a p r o t e i n on an ion-exchange column a t i t s p i can o c c u r i f i t has an uneven c h a r g e d i s t r i b u t i o n (e.g., a group o f n e g a t i v e l y charged r e s i d u e s s e p a r a t e d from t h e p o s i t i v e l y c h a r g e d r e s i d u e s ) . P e t r i l l i e t a l . u s e d an anion-exchange column f o l l o w e d by a c a t i o n - e x c h a n g e column (23), however, a s i n g l e SNAP column may a l s o be p r e p a r e d by m i x i n g anion-exchange and cation-exchange resins. The novelty of this a p p r o a c h i s t h a t by o p e r a t i n g t h e column a t t h e p i o f t h e t a r g e t


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p r o t e i n i t p a s s e s t h r o u g h t h e column u n r e t a i n e d w h i l e o t h e r p r o t e i n s are r e t a i n e d . SNAP chromatography o f f e r s s e v e r a l advantages o v e r c o n v e n t i o n a l g r a d i e n t - e l u t i o n chromatography. (1) S i n c e t h e p r o t e i n o f i n t e r e s t i s not a d s o r b e d t o t h e s u r f a c e , i t s s t r u c t u r e s h o u l d not be a l t e r e d by t h e media. T h i s means t h a t t h e p r o t e i n s h o u l d r e t a i n more a c t i v i t y . (2) The s e p a r a t i o n i s v e r y q u i c k because t h e p r o t e i n p a s s e s t h r o u g h t h e column u n r e t a i n e d . L o s s o f p r o t e i n from a d s o r p t i o n , p r o t e o l y s i s , and t i m e dependent dénaturâtion phenomena w i l l be d r a m a t i c a l l y r e d u c e d . (3) The e n t i r e chromatography column i s used d u r i n g t h e p u r i f i c a t i o n p r o c e s s . T h i s maximizes t h e e f f i c i e n c y o f t h e a d s o r b e n t and i n c r e a s e s the amount o f p r o t e i n p r o c e s s e d p e r volume o f a d s o r b e n t . (4) SNAP chromatography i s e a s i l y s c a l e d up f o r l a r g e r s e p a r a t i o n s by making t h e columns l a r g e r . (5) The pumping system i s l e s s e x p e n s i v e because s i m p l e r i s o c r a t i c systems a r e used i n l i e u o f more e x p e n s i v e g r a d i e n t pumping systems. (6) The sample i s not d i l u t e d by t h e s e p a r a t i o n p r o c e s s because i t i s p u r i f i e d by a form o f f r o n t a l chromatography. P e t r i l l i e t a l . s u g g e s t e d t h e name " I s o e l e c t r i c Chromatography" (23) f o r t h e i o n exchange approach, however, SNAP chromatography can be u s e d i n more t h a n t h e ion-exchange mode, a l b e i t w i t h a d i f f e r e n t s e p a r a t i o n mechanism. P r e l i m i n a r y r e s u l t s from S t r i n g h a m e t a l . (24,25) show t h a t tandem h y d r o p h o b i c i n t e r a c t i o n (HIC), p r o t e i n A a f f i n i t y , and s i z e - e x c l u s i o n chromatography (SEC) columns may be used i n SNAP s e p a r a t i o n s . SNAP s e p a r a t i o n s i n t h e a f f i n i t y and i m m o b i l i z e d m e t a l a f f i n i t y modes may a l s o be used. I m m u n o a f f i n i t y columns would be w e l l s u i t e d t o remove t r a c e c o n t a m i n a n t s . However, i t i s o f t e n d i f f i c u l t t o produce an a n t i b o d y t o each s i n g l e c o n t a m i n a n t . Some contaminants a r e more immunogenic t h a n o t h e r s , so most o f t h e a n t i b o d i e s would be d i r e c t e d a g a i n s t m a t e r i a l s o f h i g h immunogenicity and few o r no a n t i b o d i e s a r e produced a g a i n s t t h e r e s t . I t would be n e c e s s a r y t o i s o l a t e each contaminant and produce a n t i b o d i e s a g a i n s t it. T h i s i s not f e a s i b l e because many o f t h e c o n t a m i n a n t s a r e not even i d e n t i f i e d , much l e s s p u r i f i e d . Work r e p o r t e d by A n i c e t t i , a t Genetech (26), i n v o l v e d a c a s c a d e immunization scheme t o produce a n t i b o d i e s f o r a wide range o f a n t i g e n s . However, t h e i r work f o c u s e d on d e v e l o p i n g a n t i b o d i e s f o r a n a l y t i c a l methods t o d e t e c t t r a c e c o n t a m i n a n t s t h a t r e m a i n a f t e r p r e l i m i n a r y p u r i f i c a t i o n , not t h e p u r i f i c a t i o n i t s e l f . D e s c r i p t i o n o f t h e C o n t i n u o u s SNAP System. A f t e r a c e r t a i n amount o f use, a SNAP chromatography column will become saturated with c o n t a m i n a n t s and w i l l need t o be r e c y c l e d . To make t h e p r o c e s s c o n t i n u o u s , a second column must be used w h i l e t h e s a t u r a t e d column i s b e i n g c l e a n e d . The l o a d i n g and c l e a n i n g c y c l e i s d i v i d e d i n t o t h r e e p h a s e s . (1) The l o a d i n g phase, when sample i s b e i n g p u r i f i e d by t h e column, o c c u p i e s 50% o f t h e c y c l e t i m e . (2) D e s o r p t i o n u s i n g a 1.0 M s a l t b u f f e r r e q u i r e s 25% o f t h e c y c l e , and (3) r e - e q u i l i b r a t i o n w i t h the sample b u f f e r consumes t h e r e m a i n i n g 25% o f t h e c y c l e . Since d e s o r p t i o n and r e - e q u i l i b r a t i o n t a k e no l o n g e r t h a n l o a d i n g , two columns may be used i n p a r a l l e l f o r c o n t i n u o u s p u r i f i c a t i o n . One column i s d e s o r b e d and r e - e q u i l i b r a t e d w h i l e t h e o t h e r i s l o a d e d . The r e - e q u i l i b r a t e d column w i l l be r e a d y t o l o a d a g a i n when t h e o t h e r column becomes s a t u r a t e d . A t e n - p o r t v a l v e arrangement ( F i g u r e 2) was u s e d t o o p e r a t e t h e SNAP columns i n tandem i n a c o n t i n u o u s p u r i f i c a t i o n



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Retention Map

\

/

\

/


\ \ Cation Exchanger \ ^

/ /

Anion Exchanger

\ /

pi Buffer p H F i g u r e 1. A t pH v a l u e s below t h e p i o f t h e p r o t e i n , i t i s p o s i t i v e l y c h a r g e d and may be a d s o r b e d by c a t i o n e x c h a n g e r s , as shown by t h e i n c r e a s e i n t h e c a p a c i t y f a c t o r a t low pH. A t pH v a l u e s above t h e p i o f a p r o t e i n , i t w i l l be n e g a t i v e l y c h a r g e d and may be a d s o r b e d by a n i o n e x c h a n g e r s .

F i g u r e 2. D u a l SNAP columns were used t o p r o c e s s a c o n t i n u o u s s t r e a m o f d i l u t e d b o v i n e serum. A ten-port v a l v e switched the m o b i l e phases between serum and d e s o r p t i o n b u f f e r so t h a t as one column l o a d e d , t h e o t h e r was b e i n g r e c y c l e d .


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system. The v a l v e was automated w i t h a pneumatic a c t u a t o r c o m p u t e r i z e d LC system c o u l d r u n t h e p u r i f i c a t i o n u n a t t e n d e d .

so

a


The use o f p e r f u s a b l e (POROS) p a c k i n g m a t e r i a l s f o r t h e a d s o r b e n t a l l o w s h i g h f l o w r a t e s w i t h o u t i n c r e a s i n g back p r e s s u r e o r s l o w i n g mass t r a n s f e r (27,28). T h i s m a t e r i a l has been d e s i g n e d w i t h l a r g e t h r o u g h p o r e s t o improve mass t r a n s f e r a t h i g h f l o w r a t e s . L i n e a r v e l o c i t i e s i n e x c e s s o f 1 cm/sec on 250 mm χ 4.6 mm columns were used i n some experiments. SNAP chromatography, as w e l l as many o t h e r forms o f c o n t i n u o u s p r o t e i n separations, functions best with r e l a t i v e l y d i l u t e s o l u t i o n s ( c a . 1 mg/ml). T h i s means t h a t t h e p u r i f i e d p r o t e i n i s even more d i l u t e b e c a u s e i t o n l y r e p r e s e n t e d a f r a c t i o n o f t h e t o t a l p r o t e i n (5% would be 50 μ g / m l ) . I t i s o f t e n necessary t o concentrate the product solution by some means such as u l t r a f i l t r a t i o n . However, the s e p a r a t i o n p r o c e s s i t s e l f does not d i l u t e t h e sample, b e c a u s e t h e sample élûtes as a f r o n t a l c u r v e . An i m m o b i l i z e d m e t a l a f f i n i t y chromatography (IMAC) column was added i n tandem t o t h e SNAP columns t o adsorb and c o n c e n t r a t e t h e d i l u t e p r o d u c t p r o t e i n . IMAC (29-34) u t i l i z e s an i m m o b i l i z e d m e t a l i o n t o c h e l a t e h i s t i d i n e r e s i d u e s on p r o t e i n s . Copper I I was used b e c a u s e i t has t h e b r o a d e s t s p e c i f i c i t y f o r d i f f e r e n t p r o t e i n s (35). In t h i s way, t h e SNAP-IMAC t e c h n i q u e i s more g e n e r a l f o r p r o t e i n s o t h e r t h a n IgG. The p u r i f i e d p r o t e i n e l u t i n g from t h e SNAP columns a d s o r b s and c o n c e n t r a t e s on t h e IMAC column. The p r o t e i n i s t h e n e l u t e d from t h e IMAC column w i t h a p u l s e o f i m i d a z o l e t o d i s p l a c e t h e p r o t e i n . I m i d a z o l e does not s t r i p t h e column o f t h e m e t a l i o n , so t h e r e i s no need t o r e l o a d t h e IMAC column w i t h m e t a l . F u r t h e r , s i n c e i m i d a z o l e i s a s m a l l m o l e c u l e , i t may e a s i l y be removed, i f n e c e s s a r y , from IgG by using d i a l y s i s or a s i z e - e x c l u s i o n c a r t r i d g e . F u r t h e r p u r i f i c a t i o n , as w e l l as c o n c e n t r a t i o n , may be p e r f o r m e d on t h e IMAC column. By u s i n g a s t e p p e d g r a d i e n t o f i m i d a z o l e , t h e c o n t a m i n a n t s t h a t a r e more weakly adsorbed t o t h e IMAC column may be e l u t e d f i r s t w i t h a low c o n c e n t r a t i o n o f i m i d a z o l e . Then a h i g h e r c o n c e n t r a t i o n o f i m i d a z o l e may be used t o remove t h e c o n c e n t r a t e d p r o t e i n . F i n a l l y , a v e r y c o n c e n t r a t e d i m i d a z o l e p u l s e c o u l d be used t o remove any contaminant t h a t remained. The system used i n t h e s e s t u d i e s f a l l s i n t o t h e c a t e g o r y o f a c r o s s - c u r r e n t s i m u l a t e d moving bed and was used t o p u r i f y a s i n g l e p r o t e i n from a m i x t u r e . However, c r o s s - c u r r e n t systems can p u r i f y more t h a n one p r o t e i n s i m u l t a n e o u s l y . I f g r a d i e n t e l u t i o n were used t o d e s o r b t h e s a t u r a t e d column, a f r a c t i o n c o l l e c t o r a t t h e waste o u t l e t c o u l d c o l l e c t o t h e r p r o t e i n s as t h e y e l u t e d u r i n g t h e wash g r a d i e n t . However, p u r i f i c a t i o n o f m u l t i p l e c o n s t i t u e n t s s i m u l t a n e o u s l y may r e q u i r e t h e a d d i t i o n o f more v a l v e s and columns t o t h e system. Because two o r more p r o t e i n s a r e r a r e l y p u r i f i e d s i m u l t a n e o u s l y , no e f f o r t was given to the p u r i f i c a t i o n of m u l t i p l e p r o t e i n s . Observations. C o n c e n t r a t e d f e e d streams l o a d t h e SNAP chromatography columns f a s t e r t h a n t h e y may be desorbed, so t h e system works b e s t w i t h d i l u t e f e e d streams ( l e s s t h a n 1 mg/ml). I f t h e f e e d stream i s t o o



20

c o n c e n t r a t e d , p r o d u c t w i l l a l s o be wasted. Each t i m e t h e SNAP column i s d e s o r b e d , t h e column s t i l l c o n t a i n s t h e components o f t h e f e e d s t r e a m i n i t s v o i d volume. These components i n c l u d e t h e p r o d u c t p r o t e i n as w e l l as t h e c o n t a m i n a n t s . The p r o d u c t y i e l d , o r r e c o v e r y o f t h e SNAP chromatography system i s a f u n c t i o n o f t h e column v o i d volume (V ) and t h e volume o f f e e d s o l u t i o n t h a t p a s s e s t h r o u g h t h e column (V.) b e f o r e t h e column becomes s a t u r a t e d . Optimum r e c o v e r y (R) i s r e l a t e d t o t h e s e v a r i a b l e s by t h e f o l l o w i n g e q u a t i o n : 0


*=

1

0

0

%

To o p t i m i z e r e c o v e r y , V needs t o be maximized and V m i n i m i z e d . U s u a l l y , v e r y l i t t l e c a n be done w i t h V s i n c e i t i s a f u n c t i o n o f t h e p a c k i n g m a t e r i a l . However, t h e use o f a p a c k i n g m a t e r i a l w i t h a h i g h l o a d i n g c a p a c i t y does i n c r e a s e V . A l s o , as t h e f e e d s t r e a m i s d i l u t e d , V, i n c r e a s e s because i t t a k e s l o n g e r t o s a t u r a t e t h e column. On t h e o t h e r hand, i t t a k e s e f f o r t t o r e c o n c e n t r a t e t h e p r o d u c t p r o t e i n , so t h e r e i s a compromise between d i l u t i o n and r e c o v e r y . t

0

0

t

One way t o a v o i d t h e r e c o v e r y problem i s t o u s e two s i x - p o r t v a l v e s i n s t e a d o f one t e n - p o r t v a l v e t o c o n t r o l t h e f l o w t h r o u g h t h e SNAP columns. The f i r s t v a l v e d e t e r m i n e s which column r e c e i v e s t h e f e e d s t r e a m and t h e second v a l v e d e t e r m i n e s which column i s c o n n e c t e d to the c o l l e c t i o n v e s s e l . A f t e r t h e f i r s t v a l v e t u r n s , t h e second v a l v e i s d e l a y e d e q u i v a l e n t t o t h e t i m e i t t a k e s t h e column t o p a s s t h e v o i d volume. R e c o v e r y c o u l d always be 100% as l o n g as t h e column d i d not s a t u r a t e i n l e s s t h a n one v o i d volume. S c a l e - u p o f t h e SNAP system i s s i m p l y a m a t t e r o f i n c r e a s i n g column s i z e and f l o w r a t e s . Since the separation i s i n s e n s i t i v e t o p l a t e h e i g h t , t h e u s u a l hydrodynamic problems a s s o c i a t e d w i t h l a r g e r columns a r e r e d u c e d . T h i s i s an advantage o v e r t h o s e continuous systems t h a t r e q u i r e m u l t i p l e columns w i t h e q u i v a l e n t f l o w p r o p e r t i e s . The SNAP system o n l y r e q u i r e s t h a t t h e d e s o r p t i o n t i m e o f t h e s l o w e s t column be l e s s t h a n t h e l o a d i n g t i m e o f t h e f a s t e s t column. Columns can be matched s i m p l y by a d j u s t i n g t h e f e e d r a t e r e l a t i v e t o t h e d e s o r p t i o n flow r a t e . In t h e experiments performed i n these studies, standard a n a l y t i c a l columns (4.6 mm i . d . χ 25 cm) were o p e r a t e d a t f l o w r a t e s o f 10 ml/min. T h i s means t h a t t h e l i n e a r f l o w r a t e was i n e x c e s s o f 1 cm/second. These f l o w r a t e s were p o s s i b l e b e c a u s e a p e r f u s a b l e a d s o r b e n t (POROS) was used t o pack t h e columns. H i g h t h r o u g h p u t s were then p o s s i b l e using r a p i d m u l t i p l e c y c l e s t o perform continuous s e p a r a t i o n s (See T a b l e I ) . The a d s o r b e n t i n t h e column i s used v e r y e f f i c i e n t l y i n r a p i d m u l t i p l e c y c l e SNAP chromatography. The r a t e o f p r o t e i n p r o c e s s e d f o r a g i v e n volume o f a d s o r b e n t may be g r e a t e r t h a n 1.0 mg/min/ml o f column. The IMAC column was p l a c e d i n tandem t o t h e SNAP columns ( F i g u r e 3) t o make a c o n t i n u o u s p u r i f i c a t i o n and c o n c e n t r a t i o n system. An i n j e c t i o n v a l v e was p l a c e d between t h e SNAP columns and t h e IMAC column


2.

NADLER & REGNIER Table

I.

D u a l Column SNAP R e s u l t s Experiment 2

Experiment 3

10

5

10

1.0

0.5

1.0

Serum D i l u t i o n

1/100

1/25

1/50

Serum P r o t e i n Cone, (mg/ml)

0.781

2.13

1.09

F r a c t i o n IgG ( r e l . area 1st peak)

8.85%

6.28%

7.47%

7.8

10.7

10.9

0.94

1.28

1.30

0.061

0.047

Experiment Flow r a t e (ml/min) L i n e a r Flow Rate (cm/sec)


21


P r o c e s s i n g Rate (mg Serum/min) R a t e / C o l . Volume (mg/min/ml) IgG P r o t e i n Cone, (mg/ml)

0.084

1

-40%

Fold

4.52

12.8

12.7

48.9%

36.5%

54.8%

Purification

Approximate

Yield

>

80%

-95%

IgG P u r i t y ( r e l . a r e a 1 s t peak)

so p u l s e s o f a d e s o r p t i o n b u f f e r c o u l d be i n j e c t e d i n t o t h e IMAC column t o r e l e a s e t h e c o n c e n t r a t e d IgG. The v a l v e was f i t t e d w i t h a 2 ml sample l o o p f o r t h a t p u r p o s e . Once t h e IMAC column was s a t u r a t e d w i t h IgG, i t was e l u t e d w i t h 200 mM i m i d a z o l e t o y i e l d 95% p u r e IgG a t c o n c e n t r a t i o n s g r e a t e r t h a n 5 mg/ml. O t h e r methods o f c o n c e n t r a t i o n are a l s o p o s s i b l e . A f f i n i t y columns f o r t h e p r o t e i n may be p l a c e d a f t e r t h e SNAP columns t o c a p t u r e t h e d i l u t e p r o t e i n . Ion-exchange columns a l s o may be used, but t h e m o b i l e phase pH would have t o be a d j u s t e d so t h a t t h e p r o t e i n a c h i e v e d a net c h a r g e ( e i t h e r p o s i t i v e o r negative). IMAC was chosen because i t was s i m p l e t o use (no pH a d j u s t m e n t was n e c e s s a r y ) , and i t was f a i r l y g e n e r i c ( i t c o u l d be used f o r a number o f d i f f e r e n t p r o t e i n s because i t i s not as s p e c i f i c as a f f i n i t y columns). Some c o n t a m i n a n t s i n t h e p u r i f i e d IgG, were o b s e r v e d by SDS-PAGE. S e l e c t i v e e l u t i o n o f t h e s e c o n t a m i n a n t s was attempted i n order t o o b t a i n f u r t h e r p u r i f i c a t i o n as w e l l as c o n c e n t r a t i o n from t h e IMAC column. S a l t washes as h i g h as 1.0 M NaCl d i d not e l u t e any o f t h e IgG nor any o f t h e c o n t a m i n a n t s . The l o a d e d IMAC column was removed from t h e SNAP-IMAC system and e l u t e d w i t h a g r a d i e n t o f i m i d a z o l e . The IgG e l u t e d from t h e column a t a low i m i d a z o l e c o n c e n t r a t i o n ( l e s s t h a n 20 mM) w h i l e one contaminant e l u t e d a t 200 mM i m i d a z o l e .



22


To t a k e advantage o f t h i s s e l e c t i v e e l u t i o n w i t h o u t t h e u s e o f a linear gradient, a series of pulses of increasing imidazole b o n c e n t r a t i o n were u s e d . The i m i d a z o l e p u l s e was i n t r o d u c e d v i a t h e i n j e c t i o n v a l v e between t h e SNAP and IMAC columns. A f r a c t i o n was c o l l e c t e d a f t e r e a c h p u l s e and a n a l y z e d by SDS-PAGE ( F i g u r e 4 ) . Some high-molecular-weight c o n t a m i n a n t s a r e removed by a 1 mM i m i d a z o l e p u l s e w h i l e most o f t h e IgG e l u t e s d u r i n g t h e 10 mM and 15 mM p u l s e s . A l o w - m o l e c u l a r - w e i g h t contaminant remains u n t i l a 200 mM p u l s e i s used. By u s i n g a s e r i e s o f t h r e e i m i d a z o l e p u l s e s , 5 mM t o e l u t e t h e high-molecular-weight c o n t a m i n a n t s , 15 mM t o e l u t e t h e IgG and 200 mM t o e l u t e t h e r e m a i n i n g c o n t a m i n a n t , t h e IgG i s f u r t h e r p u r i f i e d as w e l l as c o n c e n t r a t e d on t h e IMAC column. Optimization of SNAP. O p t i m i z a t i o n o f SNAP chromatography s e p a r a t i o n s has been d i s c u s s e d i n d e t a i l (24,25). To o p t i m i z e t h e SNAP system, one must choose a s a l t c o n c e n t r a t i o n j u s t h i g h enough t o e l u t e t h e p r o t e i n of i n t e r e s t . H i g h e r s a l t c o n c e n t r a t i o n s compromise t h e s e p a r a t i o n by desorbing contaminants. To f i n d t h e optimum s a l t concentration, S t r i n g h a m e t a l . (25) s t a r t e d w i t h an e q u a t i o n d e r i v e d by Snyder e t a l . (36). The e q u a t i o n r e l a t e s i s o c r a t i c and g r a d i e n t e l u t i o n . Retention t i m e ( t ) i s r e l a t e d t o average k' i n g r a d i e n t e l u t i o n (k*), column dead volume ( t ) and t h e k' a t t h e o n s e t o f t h e g r a d i e n t (k) by: R

0

t =t R

The

0

k> l o g ( 1 ^ 2 ) C +

0

(1)

a v e r a g e k' i s g i v e n by: k*=——2—

(2)

where t i s t h e d u r a t i o n o f t h e g r a d i e n t , (Δφ) i s t h e change i n volume f r a c t i o n o f t h e e l u e n t s o l v e n t (φ i s 1.0 when t h e g r a d i e n t i s r u n from 0 t o 100%) and S i s t h e change i n l o g k' f o r t h e u n i t change i n ψ i n isocratic elution. I n i s o c r a t i c systems, r e t e n t i o n i s g i v e n by: f

log(/e')=log(Jc )-S 0

Equation

(3)

(2) may be reduced t o :

When t h e e n t i r e g r a d i e n t i s r u n from 0 t o 100%. A c a p a c i t y f a c t o r o f 10 i s i n t h e s h a r p t r a n s i t i o n range d i s c u s s e d above. S u b s t i t u t i n g t h i s v a l u e i n t o e q u a t i o n 3 and r e a r r a n g i n g g i v e s : log(ic ) = 1 + 5φ 0


(5)


NADLER & REGNIER


sample buffer

desorption buffer

F i g u r e 3. An IMAC column was added t o t h e d u a l - c o l u m n SNAP a p p a r a t u s . IgG was c o n c e n t r a t e d on t h e IMAC column and was e l u t e d with imidazole. A m i c r o p r o c e s s o r - c o n t r o l l e d pumping system was used t o automate t h e system.

F i g u r e 4. SDS-PAGE o f samples e l u t e d from t h e IMAC column a t v a r i o u s i m i d a z o l e c o n c e n t r a t i o n s (1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 200 mM). IgG p r o d u c e s two bands b e c a u s e i t i s composed o f two heavy (H) c h a i n s and two l i g h t (L) c h a i n s . M o l e c u l a r weight s t a n d a r d s a r e i n d i c a t e d on t h e l e f t (68 KDa, 48 KDa, and 12.5 KDa).



24 Substituting

(4) and

(5) i n t o e q u a t i o n

t =4?-% R

1 yields:

Clog (2 .3) +l +i*|>-log (

) ] +1

0

which s i m p l i f i e s t o : t -1.36

Continuous Purification of Proteins by Selective Nonadsorptive

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