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Chapter 11

Recovery of Recombinant Proteins by Immunoaffinity Chromatography

Downloaded by UNIV ILLINOIS URBANA-CHAMPAIGN on October 5, 2015 | http://pubs.acs.org Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch011

Pascal Bailon and Swapan K. Roy Department of Protein Biochemistry, Roche Research Center, Hoffman-La Roche, Inc., Nutley, NJ 07110

An overview of the use of immunoaffinity chromatography as a purification tool in downstream processing of therapeutically useful recombinant proteins is presented. Using the recovery process of recombinant interferon-alpha 2a as the model system, a systematic approach to the development and optimization of immunoaffinity purification systems is discussed. Advantages and problem areas of immunoaffinity chromatography are pointed out. Overall, the immunoaffinity purification method is shown to be a viable, scalable separation method for the purification of recombinant proteins. The explosion of biotechnology research and its evolution into pioneering therapeutic and diagnostic products have made great demands on the fledgling bioprocess technology. The clinical use of recombinant proteins requires the capabilities of producing biologically and biochemically homogeneous materials, reliably and economically. Immunoaffinity chromatography, based upon the specificity and reversibility of the antigen-antibody interactions, is an ideal method for the selective purification of recombinant proteins. Immunoaffinity chromatography is in fact the predecessor of affinity chromatography which became very popular in the 1970's. Most of the earlier work in this field involved the use of solidphase antigen derivatives for the purification of homologous antibodies from immune sera (1). The first well-characterized immunoadsorbent was prepared by Campbell et a l . , (2) in which the antigen ovalbumin was chemically bonded to an insoluble polymeric carrier and used for the isolation of antibodies to ovalbumin. In 1964, Gurvich and Drizlikh (3) used an immobilized antibody for the detection of radiolabeled antigens. Since the discovery of monoclonal antibody-producing hybrid cell lines by Kohler and Milstein in 1975 (4), a large number of murine monoclonal antibodies have been prepared. It is now common 0097-6156/90/0427-0150$06.00/0 © 1990 American Chemical Society

In Protein Purification; Ladisch, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


11.

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Recovery of Recombinant Proteins

p r a c t i c e to develop monoclonal a n t i b o d i e s which b i n d p r o t e i n s a t d i f f e r e n t a n t i g e n i c s i t e s and p o s s e s s d i f f e r e n t b i n d i n g a f f i n i t i e s . Consequently, the use o f m o n o c l o n a l a n t i b o d i e s i n b i o m e d i c a l i n d u s t r i e s has been i n c r e a s i n g s t e a d i l y . One such a p p l i c a t i o n i s the use o f i m m o b i l i z e d m o n o c l o n a l a n t i b o d i e s as immunoadsorbents f o r the p u r i f i c a t i o n o f b i o m o l e c u l e s such as i n t e r f e r o n s o f v a r i o u s o r i g i n s (5,6,7), i n t e r l e u k i n - 2 ( 8 ) , b l o o d c l o t t i n g f a c t o r s ( 9 ) , membrane a n t i g e n s (10) and i n t e r l e u k i n - 2 r e c e p t o r (11) among o t h e r s . The advent o f f a s t , e f f i c i e n t and c o s t - e f f e c t i v e t e c h n o l o g i e s f o r the l a r g e - s c a l e p r o d u c t i o n (12), p u r i f i c a t i o n (13) and i m m o b i l i z a t i o n (14, 15) o f monoclonal a n t i b o d i e s have made i m m u n o a f f i n i t y chromatography a f u l l - f l e d g e d t e c h n o l o g y f o r the i n d u s t r i a l s c a l e p u r i f i c a t i o n o f recombinant p r o t e i n t h e r a p e u t i c s (16). In t h i s paper we p r e s e n t an overview o f the use o f immunoaffi nity chromatography as a p u r i f i c a t i o n t o o l i n the p r o c e s s i n g o f recombinant DNA p r o t e i n s from Ε.coli and mammalian c e l l c u l t u r e s u p e r n a t a n t s . A s y s t e m a t i c a p p r o a c h to the development and o p t i m i z a t i o n o f i m m u n o a f f i n i t y p u r i f i c a t i o n systems i s p r e s e n t e d . Most o f the methodologies d e v e l o p e d f o r the i n d u s t r i a l p r o d u c t i o n o f recom b i n a n t p r o t e i n s are p r o p r i e t a r y . Consequently, we w i l l r e l y on a model system developed i n our l a b o r a t o r y , i n o r d e r to e x p l a i n the s y s t e m a t i c approach needed f o r p u r i f y i n g a recombinant p r o t e i n to homogeneity and r e n d e r i n g i t s u i t a b l e f o r t h e r a p e u t i c p u r p o s e s . Immunoaffinity

Concept

M o l e c u l a r R e c o g n i t i o n P r o p e r t y o f Monoclonal A n t i b o d i e s . Molecular r e c o g n i t i o n between a m o n o c l o n a l a n t i b o d y and i t s a n t i g e n o c c u r s through the f o r m a t i o n o f a n o n - c o v a l e n t l y bonded immunological complex. The above mentioned a n t i g e n - a n t i b o d y i n t e r a c t i o n i s the b a s i s o f i m m u n o a f f i n i t y chromatography. S i n c e most a n t i b o d i e s are b i v a l e n t , t h e o r e t i c a l l y two a n t i g e n m o l e c u l e s can b i n d to one mole c u l e o f monoclonal a n t i b o d y . However, t h i s t h e o r e t i c a l b i n d i n g c a p a c i t y i s seldom a c h i e v e d by immunoadsorbents f o r a v a r i e t y o f reasons. The i n h e r e n t p r o p e r t i e s o f a n t i g e n - a n t i b o d y r e c o g n i t i o n impart the f o l l o w i n g c h a r a c t e r i s t i c s to i m m u n o a f f i n i t y chromatography: a. Rapid f o r m a t i o n o f a s t a b l e but r e v e r s i b l e complex b. High s e l e c t i v i t y which p e r m i t s the i s o l a t i o n o f a s i n g l e molecule from a complex m i x t u r e o f components c. E q u i v a l e n t a p p l i c a b i l i t y f o r l a r g e or s m a l l m o l e c u l e s d. U s u a l l y h i g h r e c o v e r y o f b i o l o g i c a l a c t i v i t y Schematic O u t l i n e o f I m m u n o a f f i n i t y Chromatography. An o v e r a l l scheme o f i m m u n o a f f i n i t y chromatography i s g i v e n i n F i g u r e 1. Monoclonal a n t i b o d i e s c h e m i c a l l y bonded to an i n e r t polymer s u p p o r t are used as immunoadsorbents. A f t e r p r o p e r e q u i l i b r a t i o n , the crude a n t i g e n i s passed through the immunosorbent column and the unadsorbed m a t e r i a l s are washed away. The s p e c i f i c a l l y bound a n t i g e n i s then e l u t e d w i t h m i l d d e s o r b i n g agents. O p e r a t i o n a l Parameters i n I m m u n o a f f i n i t y Chromatography. In order to take f u l l advantage o f the m o l e c u l a r r e c o g n i t i o n c h a r a c t e r i s t i c s o f i m m u n o a f f i n i t y chromatography, p r o p e r d e s i g n o f the system i s o f


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utmost importance. U s i n g immunoadsorbents made f o r the p u r i f i c a t i o n o f recombinant i n t e r f e r o n as a model system, we determined the g e n e r a l o p e r a t i o n a l parameters i n the i m m u n o a f f i n i t y purification o f recombinant p r o t e i n s . S e l e c t i o n o f S u i t a b l e Monoclonal A n t i b o d i e s . The monoclonal a n t i b o d i e s chosen f o r i m m o b i l i z a t i o n s h o u l d p o s s e s s the f o l l o w i n g c h a r a c t e r i s t i c s : (1) they s h o u l d form a r e v e r s i b l e immunocomplex w i t h the d e s i r e d a n t i g e n ; and (2) they s h o u l d have h i g h enough a f f i n i t y to b i n d the a n t i g e n from even d i l u t e s o l u t i o n s , y e t low enough a f f i n i t y to a l l o w d i s s o c i a t i o n under r e l a t i v e l y m i l d c o n d i t i o n s , thus p e r m i t t i n g the r e l e a s e o f bound a n t i g e n . Determination of R e l a t i v e A f f i n i t i e s . O f t e n a n t i b o d i e s which show h i g h a f f i n i t y i n s o l i d - p h a s e immunoassays e x h i b i t l i t t l e o r no a f f i n i t y i n i m m u n o a f f i n i t y chromatography o r v i c e v e r s a (7,17). L i s t e d i n T a b l e I are the r e s u l t s o f some o f the monoclonal antibodies screened f o r immobilization. The above l i s t does not take i n t o a c c o u n t the l a r g e number o f c l o n e s s c r e e n e d a f t e r c e l l f u s i o n to i d e n t i f y the ones which produce a n t i b o d i e s s p e c i f i c f o r the a n t i g e n i c s i t e . C o m p e t i t i o n ELISA has been used to determine the r e l a t i v e a f f i n i t i e s o f monoclonal a n t i b o d i e s ( 1 7 ) . T h i s i s h e l p f u l i n n a r r o w i n g down the number o f monoclonal a n t i b o d i e s screened f o r i m m o b i l i z a t i o n . However, t h i s would not i n d i c a t e a f f i n i t i e s r e t a i n e d a f t e r i m m o b i l i z a t i o n . The most p r a c t i c a l approach i s the e m p i r i c a l method, i . e . , i m m o b i l i z a t i o n o f the monoclonal a n t i b o d y on a s m a l l s c a l e f o l l o w e d by the d e t e r m i n a t i o n o f the immunos o r b e n t ' s a n t i g e n b i n d i n g c a p a c i t y and s e l e c t i v i t y , as w e l l as i t s r e v e r s i b i l i t y with mild desorbing agents. Polymer Supports f o r

Immobilization

P h y s i c a l . C h e m i c a l and M e c h a n i c a l P r o p e r t i e s . Insoluble c a r r i e r s chosen f o r the p r e p a r a t i o n o f immunosorbents s h o u l d be: (1) i n e r t , h y d r o p h i l i c , c h e m i c a l l y s t a b l e and s h o u l d c o n t a i n an optimum number o f f u n c t i o n a l groups (e.g., h y d r o x y l , c a r b o x y l , a l i p h a t i c or a r o m a t i c p r i m a r y amines, h y d r a z i d e , e t c . ) which can be e a s i l y a c t i v a t e d f o r e f f i c i e n t a n t i b o d y c o u p l i n g (2) r i g i d beads w i t h h i g h p o r o s i t y , t h e r e b y a l l o w i n g the r a p i d passage o f p o t e n t i a l l y v i s c o u s f l u i d s a t moderate p r e s s u r e ; and (3) c a p a b l e o f p r o v i d i n g a m i c r o e n v i r o n m e n t i n terms o f b i o c o m p a t i b i l i t y and h y d r o p h i l i c i t y , which f a v o r s o p t i m a l a n t i g e n - a n t i b o d y i n t e r a c t i o n s . Flow P r o p e r t i e s o f V a r i o u s Support Media. The flow p r o p e r t i e s o f v a r i o u s column s u p p o r t media were d e t e r m i n e d ( d a t a not shown). A f t e r c a r e f u l e v a l u a t i o n we chose two NuGels ( S e p a r a t i o n I n d u s t r i e s , Metuchen, NJ) and Sepharose CL-6B (Pharmacia LKB B i o t e c h n o l o g y , Inc., Piscataway, NJ) f o r the i m m o b i l i z a t i o n o f monoc l o n a l a n t i b o d i e s . The NuGel s u p p o r t has the added advantage o f b e i n g a d u r a b l e bed support and a l l o w i n g a 4 - f o l d f l u x over agarose supports.


11.

BAILON AND ROY

Immobilization

153


Methods

Common S t r a t e g y . The common s t r a t e g y f o r i m m o b i l i z a t i o n i n v o l v e s treatment of the polymer s u p p o r t w i t h a reagent or sequence o f reagents to c o n v e r t i t i n t o a c h e m i c a l l y r e a c t i v e form. The p r o t e i n i s then a l l o w e d to r e a c t w i t h the s u p p o r t so t h a t s t a b l e c o v a l e n t bonds are formed. Numerous i m m o b i l i z a t i o n methods have been r e p o r t e d i n the l i t e r a t u r e (14,15).


Activation

Procedures

We chose the f o l l o w i n g f o u r a c t i v a t i o n p r o c e d u r e s f o r p r e p a r a t i o n o f immunosorbents.

the

N-Hydroxysuccinimide E s t e r (NHS) D e r i v a t i v e s . An N-hydroxysuccinimide e s t e r d e r i v a t i v e of c r o s s l i n k e d agarose having the formula Agarose-OCH CH(OH)CH NHCOCH2CH CONHS 2

2

2

has been p r e p a r e d a c c o r d i n g to the p u b l i s h e d p r o c e d u r e s (18,19,20,). The c o m m e r c i a l l y a v a i l a b l e NHS e s t e r d e r i v a t i v e o f NuGel (NuGel P-AF P o l y - N - H y d r o x y s u c c i n i m i d e , S e p a r a t i o n I n d u s t r i e s , Metuchen, NJ) has the f o l l o w i n g s t r u c t u r e . N u G e l - ( C H ) 0CH CH NHCH C0NHS 2

3

2

2

2

C a r b o n y l - i m i d a z o l e (CI) D e r i v a t i v e s . The i m i d a z o l y l - c a r b a m a t e d e r i v a t i v e s o f c r o s s - l i n k e d agarose and NuGel were p r e p a r e d a c c o r d i n g to the p r o c e d u r e o f B e t h e l e t a l . (21). The Aldehyde (CHO) D e r i v a t i v e s . C r o s s l i n k e d agarose and NuGel s u b j e c t e d to p e r i o d a t e o x i d a t i o n to c o n v e r t them i n t o t h e i r r e s p e c t i v e aldehyde d e r i v a t i v e s (22,23). These d e r i v a t i v e s r e a d i l y r e a c t w i t h amine n u c l e o p h i l e s to y i e l d c o v a l e n t l y c o u p l e d l i g a n d i n aqueous s o l u t i o n s .

are

Hydrazide D e r i v a t i v e . P e r i o d a t e o x i d i z e d monoclonal a n t i b o d y i s c o u p l e d to the c o m m e r c i a l l y a v a i l a b l e a d i p i c d i h y d r a z i d e d e r i v a t i v e o f NuGel ( S e p a r a t i o n I n d u s t r y , Metuchen, NJ) (24). T h e o r e t i c a l l y , o r i e n t e d c o u p l i n g can be a c h i e v e d by c o u p l i n g through the sugar m o i e t i e s of the Fc r e g i o n o f the IgG m o l e c u l e . T h i s , i n t u r n , s h o u l d r e s u l t i n h i g h e r a n t i g e n b i n d i n g c a p a c i t y than t h a t a c h i e v e d f o r immunosorbents p r e p a r e d by c o u p l i n g v i a the p r i m a r y amino groups (see T a b l e I V ) . Immobilization Procedure. The a c t i v a t e d g e l i s q u i c k l y washed w i t h three volumes o f i c e - c o l d water i n a c o a r s e s i n t e r e d g l a s s f u n n e l . The g e l i s then mixed w i t h an equal volume o f p r o t e i n s o l u t i o n (monoclonal a n t i b o d y o f known c o n c e n t r a t i o n ) made up i n the coupl i n g b u f f e r (0.1M p o t a s s i u m phosphate c o n t a i n i n g 0.1M NaCl, pH, 7.0) i n an a p p r o p r i a t e v e s s e l and shaken f o r 4-16 hours a t 4°C. The unbound p r o t e i n i s c o l l e c t e d by f i l t e r i n g the r e a c t i o n m i x t u r e . The



154


g e l i s washed with two volumes o f phosphate b u f f e r e d s a l i n e (PBS), pH, 7.4. The f i l t r a t e and washes a r e combined and a s m a l l a l i q u o t i s d i a l y z e d a g a i n s t PBS. The g e l i s then t r e a t e d w i t h e t h a n o l a m i n e - H C l , pH, 7.0 t o n e u t r a l i z e any r e m a i n i n g a c t i v a t e d groups. I t i s washed w i t h PBS and s t o r e d as a s u s p e n s i o n i n PBS i n the p r e s e n c e o f 0.1% sodium a z i d e , a t 4°C. The volume o f the unbound p r o t e i n s o l u t i o n i s r e c o r d e d and the p r o t e i n c o n c e n t r a t i o n i s d e t e r m i n e d from the d i a l y z e d a l i q u o t by the method o f Lowry e t a l (25). From these two v a l u e s t h e t o t a l unbound p r o t e i n i s c a l c u l a t e d . The d i f f e r e n c e between the s t a r t i n g amount and u n c o u p l e d antibody i n the p o o l e d f i l t r a t e and washes, d i v i d e d by the g e l volume g i v e s the a n t i b o d y c o u p l i n g d e n s i t y (mg/ml g e l ) . F a c t o r s A f f e c t i n g C o u p l i n g E f f i c i e n c y and R e s i d u a l Immunoreacti v i t y . Factors a f f e c t i n g the c o v a l e n t i m m o b i l i z a t i o n o f p o l y c l o n a l a n t i b o d i e s have been s t u d i e d e x t e n s i v e l y by Comoglio e t a l . ( 2 6 ) . C o u p l i n g c o n d i t i o n s such as pH, a c t i v a t e d group d e n s i t y on the m a t r i x and the amount o f a n t i b o d y c o u p l e d p e r u n i t volume o f g e l ( c o u p l i n g density) i n f l u e n c e the l o s s o f immunoreactivity usually accompanied by i m m o b i l i z a t i o n . R e s i d u a l Immunoreactivity. The amount o f a n t i g e n b i n d i n g c a p a c i t y r e t a i n e d by the monoclonal a n t i b o d y a f t e r i m m o b i l i z a t i o n i s d e f i n e d as the r e s i d u a l i m m u n o r e a c t i v i t y o f the immunoadsorbent. T h i s c a n be d e t e r m i n e d e x p e r i m e n t a l l y . A s m a l l immunosorbent column o f known volume (0.5-1.0 ml) i s s a t u r a t e d w i t h an e x c e s s o f a n t i g e n ( p u r i f i e d o r crude) , the unadsorbed m a t e r i a l s a r e washed away and t h e s p e c i f i c a l l y bound a n t i g e n i s e l u t e d w i t h m i l d d e s o r b i n g agents. From the p r o t e i n c o n t e n t o f t h e bound a n t i g e n and i t s M , the number o f nanomoles o f the a n t i g e n bound i s c a l c u l a t e d . The number o f nanomoles o f a n t i g e n bound p e r u n i t volume o f g e l i s taken as the a n t i g e n b i n d i n g c a p a c i t y o f the immunosorbent. r

E f f e c t o f c o u p l i n g pH. A t a c o n s t a n t r a t i o o f a n t i b o d y to a c t i v a t e d matrix (15 mg/ml g e l ) the c o u p l i n g e f f i c i e n c i e s and r e s i d u a l i m m u n o r e a c t i v i t i e s o f t h e immunosorbents d e r i v e d from NHS-ester and aldehyde d e r i v a t i v e s were s t u d i e d . R e s u l t s are i l l u s t r a t e d i n F i g u r e 2. At lower pHs the c o u p l i n g i s e s s e n t i a l l y through the low pKa alpha-amino groups, whereas a t h i g h e r pHs the h i g h pKa e p s i l o n - a m i n o groups o f t h e l y s i n e s o f the IgG m o l e c u l e a r e a l s o a v a i l a b l e f o r c o u p l i n g , which a c c o u n t s f o r the i n c r e a s e d c o u p l i n g e f f i c i e n c i e s . The r e s i d u a l i m m u n o r e a c t i v i t y i s optimum a t pH, 6-7. At t h i s pH fewer amino groups a r e n o n - p r o t o n a t e d and c o n s e q u e n t l y , fewer m u l t i p o i n t attachments o c c u r . T h i s s h o u l d r e s u l t i n l e s s s t e r i c h i n d r a n c e t o the a n t i b o d y , which i n t u r n s h o u l d a l l o w g r e a t e r a c c e s s i b i l i t y o f the a n t i g e n t o the a n t i b o d y b i n d i n g s i t e s . When the a c t i v a t e d groups a r e s t a b l e , as i n the C H O - d e r i v a t i v e s , the pH o f the c o u p l i n g r e a c t i o n i s n o t as important as i n the l a b i l e NHS-ester d e r i v a t i v e s . E f f e c t o f A c t i v a t e d Group D e n s i t y . The e f f e c t o f f u n c t i o n a l group d e n s i t y on the m a t r i x i s summarized i n T a b l e I I . A t v e r y low f u n c t i o n a l group d e n s i t y (2-5 μ moles/ml g e l ) c o u p l i n g e f f i c i e n c y i s v e r y poor and the a n t i g e n b i n d i n g c a p a c i t y



11. BAILON AND ROY

F i g u r e 1. A Schematic I l l u s t r a t i o n o f I m m u n o a f f i n i t y

Table I.

Antibodies Screened Which B i n d The A n t i g e n

Anticen r I F N - a l p h a A (Roche) r I F N - b e t a (7) r I L - 2 (Roche) U r o k i n a s e ( P u r i f i c a t i o n Eng.)

II. Effect

A c t i v a t e d Groups ^moles/ml g e l 2-5 20-40 100-200

Chromatography.

Results of Monoclonal A n t i b o d i e s Screened f o r I m m o b i l i z a t i o n

No.

Table

155


4 2 1 1

13 5 6 2

o f F u n c t i o n a l Group

Coupling

No. A n t i b o d i e s S u i t a b l e For Immunoaffinity Chromatography

Efficiency

Poor Optimum High

Density

Immunoreactivity

High Optimum Poor


156



i s h i g h . At the medium d e n s i t y (20-40 μ moles/ml g e l ) b o t h t h e c o u p l i n g e f f i c i e n c y and the r e s i d u a l i m m u n o r e a c t i v i t y a r e o p t i m a l . At r e l a t i v e l y h i g h f u n c t i o n a l group d e n s i t y (100-200 μ moles/ml g e l ) c o u p l i n g e f f i c i e n c y i s the h i g h e s t b u t t h e r e i s s u b s t a n t i a l l o s s i n i m m u n o r e a c t i v i t y , p o s s i b l y due t o m u l t i p o i n t attachment. With the i m i d a z o l y l - c a r b a m a t e - d e r i v a t i v e s o f agarose, 40-50 μ moles o f a c t i v a t e d groups/ml g e l were needed f o r e f f i c i e n t c o u p l i n g ( d a t a not shown). T h i s may be due to the l a c k o f a s p a c e r arm between the m a t r i x and the IgG m o l e c u l e . E f f e c t o f Antibody Coupling Density. When the a c t i v a t e d m a t r i x i s l a b i l e as i n the N H S - d e r i v a t i v e , c o n t a c t r a t i o s o f p r o t e i n (mg) t o s u p p o r t (ml g e l ) determine the c o u p l i n g e f f i c i e n c y . When the a n t i b o d y c o n c e n t r a t i o n s ranged from 1-20 mg/ml g e l , >90% c o u p l i n g e f f i c i e n c y was a c h i e v e d . The r e s i d u a l i m m u n o r e a c t i v i t i e s a t v a r i o u s a n t i b o d y l o a d i n g s on the m a t r i x a r e summarized i n T a b l e I I I . In accordance w i t h the o b s e r v a t i o n o f o t h e r s , (26) h i g h a n t i b o d y l o a d i n g s r e s u l t e d i n lower r e s i d u a l i m m u n o r e a c t i v i t i e s , p o s s i b l y due t o s t e r i c h i n d r a n c e . Residual

Immunoreactivities

of Various

Immunosorbents

The i m m u n o r e a c t i v i t i e s r e t a i n e d by the immunosorbents depend n o t o n l y upon the f a c t o r s d i s c u s s e d p r e v i o u s l y b u t a l s o upon the c h e m i c a l n a t u r e o f the a c t i v a t e d m a t r i c e s used. T a b l e IV l i s t s t h e v a r i o u s immunosorbents w i t h r e s p e c t t o t h e i r r e s i d u a l immunoreacti v i t i e s . F o r p r a c t i c a l reasons, the a n t i b o d y l o a d i n g was chosen a t 8-15 mg/ml g e l . A t t h i s a n t i b o d y d e n s i t y a p p r o x i m a t e l y 1 mg recombinant p r o t e i n ( i n t e r f e r o n - a l p h a 2a) c a n be p u r i f i e d p e r ml g e l . N e u t r a l pH was chosen f o r a n t i b o d y c o u p l i n g . Among the immunosorbents s t u d i e d , the a d i p i c d i h y d r a z i d e d e r i v a t i v e had the b e s t a n t i g e n b i n d i n g c a p a c i t y due t o the o r i e n t e d c o u p l i n g through the c a r b o h y d r a t e m o i e t i e s o f the Fc domain o f t h e monoclonal a n t i b o d y . S i n c e the N H S - d e r i v a t i v e s o f agarose and NuGel were r e a d i l y a v a i l a b l e , we used them f o r a l l o f our s t u d i e s . The NuGel immunosorbent has the added advantages o f b e i n g a d u r a b l e b e d s u p p o r t and a l l o w i n g a f o u r - f o l d i n c r e a s e i n f l u x (23) as compared to the c r o s s - l i n k e d agarose. These a r e i m p o r t a n t f a c t o r s t o c o n s i der when s c a l i n g up the p r o d u c t i o n o f recombinant p r o t e i n s . D e t e c t i o n and P r e v e n t i o n o f A n t i b o d y

Leaching

from

Immunosorbents

The c o v a l e n t bond formed between the a n t i b o d y and the m a t r i x d u r i n g i m m o b i l i z a t i o n may n o t be c o m p l e t e l y s t a b l e . T r a c e amounts o f i m m o b i l i z e d a n t i b o d y may l e a c h from the column d u r i n g the immuno a f f i n i t y p u r i f i c a t i o n p r o c e s s , thereby c o n t a m i n a t i n g the f i n a l b u l k product. D e t e c t i o n Method. A s e n s i t i v e , n o n - c o m p e t i t i v e , sandwich ELISA i s used t o d e t e c t a n t i b o d y l e a c h i n g from the immunosorbent d u r i n g column o p e r a t i o n s ( d a t a n o t shown). The lower l i m i t o f the a s s a y ' s s e n s i t i v i t y i s 0.1 ng/ml. S t a b i l i z a t i o n o f Immobilized Monoclonal A n t i b o d i e s . The use o f g l u t a r a l d e h y d e c r o s s l i n k i n g to prevent immobilized p r o t e i n leakage has been r e p o r t e d p r e v i o u s l y ( 2 7 ) . We have s u c c e s s f u l l y used t h i s


11.

BAILON AND ROY

157


100


80

c Φ υ

60

»_ Φ

û.

40

20

ο % Recovery of Immunoreactivity Following NHS Coupling -

• % Recovery of Immunoreactivity Following CMO Coupling • NHS Coupling Efficiency • CHO Coupling Efficiency I I I 10 Coupling pH

F i g u r e 2. E f f e c t o f pH on C o u p l i n g E f f i c i e n c y and I m m u n o r e a c t i v i t y .

Table I I I .

Ab C o u p l e d (mg/ml) 0.6 3.1 6.2 11.7 17.7 24.0

E f f e c t o f Antibody

Binding Capacity (mg/ml) 0.13 0.45 0.76 1.43 1.86 1.76

Concentration

R e s i d u a l Immunoreactivity Expected Observed Recovery (nmoles/ml) (%) 8 39 78 148 224 304

7 23 40 74 97 92

89 60 51 50 43 30

Monoclonal antibody to i n t e r f e r o n - a l p h a A a t v a r i o u s concentra t i o n s (1-25 mg/ml) was i m m o b i l i z e d on NuGel-NHS e s t e r d e r i v a t i v e a c c o r d i n g t o the p r o c e d u r e d e s c r i b e d i n t h e t e x t . I m m u n o r e a c t i v i t i e s a r e c a l c u l a t e d t a k i n g i n t o a c c o u n t t h e two b i n d i n g s i t e s o f IgG p e r m o l e c u l e and t h e m o l e c u l a r w e i g h t o f 158 Kd. M o l e c u l a r weight o f r I F N - a l p h a 2a i s t a k e n as 19.2 Kd f o r the c a l c u l a t i o n o f observed i m m u n o r e a c t i v i t i e s .


158


t e c h n i q u e to s t a b i l i z e the c o v a l e n t l y bonded a n t i b o d y and the r e s u l t s are summarized i n T a b l e V. At low c o n c e n t r a t i o n s o f g l u t a r a l d e h y d e and under c o n t r o l l e d c o n t a c t time the a n t i b o d y l e a c h i n g from immunosorbents was r e d u c e d to n o n - d e t e c t a b l e l e v e l s , w i t h o u t s i g n i f i c a n t l o s s i n immunoreactivities .


F a s t Assays

f o r M o n i t o r i n g Downstream P u r i f i c a t i o n

Steps

The s u c c e s s o f l a r g e - s c a l e p u r i f i c a t i o n p r o c e s s e s depends upon the a v a i l a b i l i t y o f r a p i d and r e l i a b l e a s s a y s f o r m o n i t o r i n g the downstream p u r i f i c a t i o n s t e p s . B i o a s s a y s and ELISAs a r e time consuming and o f t e n g i v e ambiguous r e s u l t s , e s p e c i a l l y i n the e a r l y s t a g e s o f the p u r i f i c a t i o n scheme. Roy e t a l (28) r e p o r t e d the use o f an automated h i g h - p e r f o r m a n c e immunosorbent a s s a y f o r m o n i t o r i n g recombinant l e u k o c y t e A i n t e r f e r o n p u r i f i c a t i o n s t e p s . S i m i l a r immunoa d s o r b e n t a s s a y s are found to be a f a s t and s i m p l e way o f m o n i t o r i n g the downstream p u r i f i c a t i o n o f o t h e r recombinant p r o t e i n s . Immunoaffinity

Purification

Procedures

In t h i s s e c t i o n , we f o c u s our a t t e n t i o n on the p r o c e d u r e s i n v o l v e d i n the i m m u n o a f f i n i t y p u r i f i c a t i o n o f c l i n i c a l grade recombinant p r o t e i n s from c o l i and c u l t u r e d c e l l s u p e r n a t a n t s . T h e o r e t i c a l a s p e c t s o f the f a c t o r s i n v o l v e d i n the l a r g e - s c a l e i m m u n o a f f i n i t y p u r i f i c a t i o n p r o c e s s have been r e v i e w e d by Chase ( 2 9 ) . In h i s review a r t i c l e , Sharma (30) has p r e s e n t e d an e x c e l l e n t overview o f the r e c o v e r y o f recombinant p r o t e i n s from E . c o l i . Use o f immunoa f f i n i t y chromatography i n the p u r i f i c a t i o n o f b i o m o l e c u l e s from c u l t u r e d c e l l s i s d e s c r i b e d by B o s c h e t t i e t a l ( 3 1 ) . G e n e r a l p u r i f i c a t i o n schemes f o r the p r o d u c t i o n o f p h a r m a c e u t i c a l grade recombinant p r o t e i n s from m i c r o b i a l and mammalian s o u r c e s are g i v e n below i n F i g u r e 3. S o l u b i l i z a t i o n and R e n a t u r a t i o n o f Recombinant P r o t e i n s E x t r a c t i o n o f the d e s i r e d p r o t e i n from JL_ c o l i i n i t s n a t i v e form poses unique problems, e s p e c i a l l y when the p r o t e i n i s e x p r e s s e d i n h i g h c o n c e n t r a t i o n s i n an i n s o l u b l e form w i t h i n the i n c l u s i o n b o d i e s . The c e l l s a r e d i s r u p t e d by m e c h a n i c a l , enzymatic or chemic a l means. For example, i n t e r f e r o n i s e x t r a c t e d by s i m p l y s t i r r i n g the f r o z e n and thawed c e l l s i n b u f f e r s c o n t a i n i n g dénaturants l i k e g u a n i d i n e h y d r o c h l o r i d e (GuHCl) and n o n - i o n i c d e t e r g e n t s such as T r i t o n X-100, Tween-20, e t c . Recombinant i n t e r l e u k i n - 1 i s s o l u b i l i z e d by s i m p l e e x t r a c t i o n a f t e r h o m o g e n i z a t i o n . S i n c e recombinant i n t e r l e u k i n - 2 ( r I L - 2 ) i s e x p r e s s e d i n JL_ c o l i i n h i g h c o n c e n t r a t i o n s w i t h i n the i n c l u s i o n b o d i e s , s p e c i a l t r e a t m e n t s a r e required. These i n c l u d e h o m o g e n i z a t i o n , i s o l a t i o n o f i n c l u s i o n b o d i e s , washing the i n c l u s i o n b o d i e s to remove unwanted c e l l u l a r p r o t e i n s , s o l u b i l i z i n g the r I L - 2 w i t h s t r o n g dénaturants such as 7M GuHCl and f i n a l l y , d i l u t i n g the e x t r a c t and g i v i n g the p r o t e i n enough time to r e f o l d . S t u d i e s c o n d u c t e d by L i g h t (32) have shown t h a t o p t i m a l r e f o l d i n g o c c u r s when the p r o t e i n c o n c e n t r a t i o n i s a t o r below the micromolar range. Consequently, r e l a t i v e l y large d i l u t i o n s o f the d e n a t u r e d e x t r a c t s , f o l l o w e d by a g i n g f o r v a r i o u s


11.

BAILON AND ROY

Recovery ofRecombinant Proteins

T a b l e IV. R e s i d u a l I m m u n o r e a c t i v i t i e s


Immunosorbents Ab Coupled (mg/ml) Agarose-NHS NuGel-NHS Agarose-CI NuGel-CI Agarose-CHO NuGel-CHO NuGel-Hydrazide

Immunoreactivities

o f V a r i o u s Immunosorbents

B i n d i n g Capac. (mg/ml)

Residual Immunoreactivity Recovery Observed Expected (nmoles/ml) (%)

a r e c a l c u l a t e d as i n T a b l e I I I .

T a b l e V. D e t e c t i o n and P r e v e n t i o n o f A n t i b o d y

Glutaraldehyde % (v/v) 0 0 0 1

* **

50 50 34 25 41 48 73

74 70 54 40 77 76 77

148 140 157 158 186 157 106

1.43 1.34 1.03 0.77 1.48 1.45 1.47

11.7 11.1 12.4 12.5 14.7 12.4 8.4

159

0 1 5 0

Leaching

Agarose-NHS ng/ml* %**

Agarose-CHO ng/ml %**

8 2 0 0

10 2 0 0

100 97 96 96

2 2 5 1

1 7 0 0

100 99 96 95

Antibody l e a c h i n g Residual Immunoreactivities Immunoadsorbents were t r e a t e d w i t h b i f u n c t i o n a l f o l l o w e d by N a B H ^ r e d u c t i o n .

glutaraldehyde

I E.coli Cells I Cell Membrane Preparation Extraction, Dilution and Concentration/Diafiltration

Cell Culture Supernatant

Immunoadsorbent Column 1

Gel Filtration

j

Concentration/Diafiltration

ι

~~

Bulking F i g u r e 3. I m m u n o a f f i n i t y

P u r i f i c a t i o n Schemes f o r Recombinant P r o t e i n s



160


p e r i o d s o f time are r e q u i r e d . The r e s u l t s o f such an a g i n g o r r e f o l d i n g experiment c o n d u c t e d w i t h 7M GuHCl e x t r a c t e d r I L - 2 a f t e r 4 0 - f o l d d i l u t i o n w i t h PBS i s g i v e n i n T a b l e V I . In some i n s t a n c e s , the i n c l u s i o n o f r e d u c i n g agents l i k e DTT i n the e x t r a c t i o n b u f f e r i s h e l p f u l i n s o l u b i l i z i n g the recombinant p r o t e i n s . When r e d u c i n g a g e n t s a r e used i n t h e e x t r a c t i o n b u f f e r , care s h o u l d be taken t o lower the c o n c e n t r a t i o n o f the r e d u c i n g agents so t h a t they w i l l n o t a d v e r s e l y a f f e c t the a f f i n i t y adsorbents which c o n t a i n d i s u l f i d e bonds; f o r example a n t i b o d y o r r e c e p t o r adsorbent columns. I n g e n e r a l , each recombinant p r o t e i n may r e q u i r e customized e x t r a c t i o n p r o c e d u r e s . U s u a l l y no s p e c i a l treatments a r e needed f o r the c e l l c u l t u r e s u p e r n a t a n t s and they can be a p p l i e d to the a d s o r b e n t column a f t e r a s i m p l e f i l t r a t i o n step. Adsorption A d s o r p t i o n i s one o f the most c r i t i c a l a s p e c t s o f i m m u n o a f f i n i t y chromatography. D u r i n g a d s o r p t i o n , the crude m a t e r i a l i s k e p t i n a b u f f e r which a l l o w s maximum a d s o r p t i o n . I n o r d e r t o e n s u r e t h a t no p r o d u c t i s wasted d u r i n g the a d s o r p t i o n phase, s u f f i c i e n t c o n t a c t time between the s o l u b l e a n t i g e n and the immunosorbent i s m a i n t a i n e d by c a r e f u l l y c h o o s i n g the f l o w r a t e . Washing The purpose o f washing t h e immunosorbent immediately after adsorption i s two-fold: (1) t o remove the c r u d e m a t e r i a l s from w i t h i n o r s u r r o u n d i n g the immunosorbent beads and (2) t o remove m a t e r i a l s n o n - s p e c i f i c a l l y bound e i t h e r t o the s u p p o r t o r t o the i m m o b i l i z e d a n t i b o d y . N o n - s p e c i f i c b i n d i n g t o the s u p p o r t c a n be m i n i m i z e d , b u t i s r a r e l y e l i m i n a t e d c o m p l e t e l y . E l e c t r o s t a t i c as w e l l as hydrophobic i n t e r a c t i o n s between the IgG m o l e c u l e and extraneous m a t e r i a l s i n the crude e x t r a c t a r e another s o u r c e o f n o n - s p e c i f i c b i n d i n g . These n o n - s p e c i f i c a l l y bound c o n t a m i n a n t s c a n u s u a l l y be reduced t o low l e v e l s by washing e x t e n s i v e l y w i t h b u f f e r s c o n t a i n i n g s a l t s a t n e u t r a l o r s l i g h t l y a l k a l i n e pH o r by i n c l u s i o n o f low c o n c e n t r a t i o n s o f n o n - i o n i c d e t e r g e n t s i n the s t a r t i n g m a t e r i a l s and i n a l l b u f f e r s used f o r washing. Elution The e l u t i o n o f adsorbed a n t i g e n from the immunosorbent i s a c h i e v e d by c a u s i n g the d i s s o c i a t i o n o f the a n t i g e n - a n t i b o d y complex. N o n - s p e c i f i c e l u t i o n methods a r e commonly used f o r the d e s o r p t i o n o f a n t i g e n s . These e l u e n t s i n v o l v e low o r h i g h pH b u f f e r s , p r o t e i n dénaturants such as u r e a o r GuHCl and c h a o t r o p i c agents l i k e potassium t h i o c y a n a t e . I f the a n t i g e n i n v o l v e d i s s t a b l e a t a c i d i c pH and i s r e a d i l y e l u t e d from the immunosorbent under t h e s e c o n d i t i o n s , an e l u e n t o f c h o i c e i s a low pH (

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