Electron Paramagnetic Resonance Spectroscopy Studies of


Jul 23, 2009 - Electron Paramagnetic Resonance Spectroscopy Studies of Immobilized Monoclonal Antibody Structure and Function. Erik J. Fernandez ...
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15 Electron Paramagnetic Resonance Spectroscopy Studies of Immobilized Monoclonal Antibody Structure and Function 1

Erik J. Fernandez, Forrest B. Fernandez, Roger B. Jagoda, and Douglas S. Clark

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School of Chemical Engineering, Cornell University, Ithaca, NY 14853

Electron paramagnetic resonance (EPR) spectra were taken of two spin labeled haptens combined with soluble and immobilized anti-2,4-dinitrophenyl (anti-DNP) IgG2b and IgE antibodies. Different association constants between soluble antibodies and spin labeled haptens were accompanied by differences in maximum peak-to-peak splittings in EPR spectra. When the IgG2b antibodies were immobilized using CNBr-activated Sepharose and immobilized Protein-A, lowered specific activities were observed (1.2 for CNBr-Sepharose at .58 mg antibody/ml gel, 0.8 for Protein-Α Sepharose at 1.7 mg antibody/ml gel), and the binding constant of CNBr Sepharose-immobilized antibody was 44% that of the soluble antibody. Furthermore, the EPR spectrum of spin label combined with IgG2b changed measurably upon immobilization of the antibody to CNBr-Sepharose. A f f i n i t y chromatography u t i l i z i n g monoclonal a n t i b o d i e s immobilized t o a s o l i d m a t r i x h a s been used a s a p u r i f i c a t i o n method f o r s e v e r a l years w i t h c h a r a c t e r i s t i c advantages and disadvantages.(1-3) A n t i b o d i e s are p r o t e i n s produced by v e r t e b r a t e s "designed" t o b i n d s p e c i f i c molecules, r e f e r r e d t o as antigens. When u t i l i z e d i n t h e laboratory o r production f a c i l i t y , antibodies immobilized t o a s u i t ­ a b l e s u p p o r t a l l o w a n a n t i g e n t o b e s e p a r a t e d f r o m many o t h e r components o f a b i o c h e m i c a l p r o c e s s stream. The s t r o n g i n t e r a c t i o n between antibody and a n t i g e n ( w i t h e q u i l i b r i u m c o n s t a n t s as h i g h a s 10 M ) (4)c a nbe a great advantage i n product recovery from f e r m e n t a t i o n b r o t h s , where product c o n c e n t r a t i o n s c a nbe as l o w a s 10 g/i i n e x t r e m e c a s e s . ( 5 ) I t c a n a l s o b e a d i s a d v a n t a g e , how­ e v e r , s i n c e p o t e n t i a l l y d e n a t u r i n g c o n d i t i o n s may b e r e q u i r e d t o 1 4

1

6

1

To whom correspondence should be addressed 0097-6156/ 86/ 0314-0208506.00/ 0 © 1986 American Chemical Society

Asenjo and Hong; Separation, Recovery, and Purification in Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

15.

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Ε PR

Spectroscopy of Monoclonal Antibodies

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c a u s e d i s s o c i a t i o n o f a t i g h t l y bound a n t i g e n . Another disadvan­ tage o f immunoaffinity chromatography i s the c o s t . Monoclonal a n t i b o d i e s purchased i n l a r g e q u a n t i t i e s from s p e c i a l i z e d companies now s e l l f o r a b o u t $ 2 0 0 0 / g , a l t h o u g h t h i s n u m b e r i s e x p e c t e d t o d r o p s i g n i f i c a n t l y a s more c o s t e f f e c t i v e p r o d u c t i o n methods a r e developed. Nonetheless, a wide v a r i e t y o f b i o l o g i c a l products have been p u r i f i e d u s i n g monoclonal a n t i b o d i e s , a t l e a s t i n the l a b o r a ­ tory. (For a n o v e r v i e w , see r e f e r e n c e 6 ) . In order to maximize the u t i l i t y o f immobilized monoclonal a n t i b o d i e s and m i n i m i z e c o s t s when t h e y a r e u s e d , i t i s n e c e s s a r y t o have a m o l e c u l a r - l e v e l understanding o f the e f f e c t s o f i m m o b i l i z a ­ t i o n on a n t i b o d i e s used i n the p r e p a r a t i o n o f immunosorbents. S e v e r a l s t u d i e s h a v e a l r e a d y shown t h a t i m m o b i l i z a t i o n c a n h a v e a s i g n i f i c a n t e f f e c t on antibody a c t i v i t y . F o r e x a m p l e , B o l t o n and H u n t e r (7) f o u n d t h a t t h e t o t a l a c t i v i t y o f a n t i - h u m a n g r o w t h hormone a n t i b o d i e s f e l l when c o u p l e d t o S e p h a r o s e and c e l l u l o s e supports. I n a d d i t i o n , they o b s e r v e d a drop i n what they c a l l e d " s e n s i t i v i t y " , a q u a n t i t y i n v e r s e l y r e l a t e d t o the b i n d i n g s t r e n g t h of the antibody. I n a r e l a t e d s t u d y , E v e l e i g h a n d L e v y (8) s t u d i e d the e f f e c t o f immobilized antibody l o a d i n g on s p e c i f i c a c t i v i t y , t h a t i s , t h e number o f a n t i g e n s t h a t c o u l d b i n d t o a n a n t i b o d y molecule. They found t h a t a s the l o a d i n g o f a n t i b o d y o n the s u p p o r t w a s i n c r e a s e d , t h e o v e r a l l b i n d i n g c a p a c i t y o f t h e immuno­ sorbent i n c r e a s e d , but the s p e c i f i c a c t i v i t y o f i m m o b i l i z e d a n t i ­ body dropped. A s a f i n a l example c o n s i d e r the r e s u l t s o f Weston a n d S c o r e r , who f o u n d t h a t a s t h e l o a d i n g o f I g G a n t i b o d y o n S e p h a r o s e was i n c r e a s e d , t h e s p e c i f i c a c t i v i t y o f t h e a n t i b o d i e s d e c r e a s e d t o t h e e x t e n t t h a t a maximum i n t o t a l b i n d i n g c a p a c i t y was o b s e r v e d . ( 9 ) Before p o s t u l a t i n g probable causes o f t h i s b e h a v i o r , i t i s h e l p f u l t o r e v i e w some o f t h e g e n e r a l c h a r a c t e r i s ­ tics of antibodies. A n t i b o d i e s , o r immunoglobulins, are globular p r o t e i n s with m o l e c u l a r w e i g h t s o f 1 5 0 t o 200 k d a l . T h e y a r e made u p o f f o u r s u b u n i t s , t w o " h e a v y " and t w o " l i g h t " c h a i n s , w h i c h t o g e t h e r f o r m a "Y"-shaped molecule. The stem o f the antibody i s r e f e r r e d t o a s t h e " c o n s t a n t " o r F ^ r e g i o n , w h i l e t h e two b r a n c h e s c o n t a i n i n g t h e combining s i t e s

are

called

F , fragments. N o t a b l y , these two ab r e g i o n s a r e j o i n e d b y few c o v a l e n t b o n d s , g i v i n g t h e m o l e c u l e a great deal o fmotional flexibility. With these s t r u c t u r a l features i n mind, there are s e v e r a l p o s s i b l e c a u s e s one m i g h t p u t f o r w a r d a s r e s p o n s i b l e f o r t h e reduced s p e c i f i c a c t i v i t y o f antibodies on a support, as depicted s c h e m a t i c a l l y i n F i g u r e 1. F i r s t , a p o r t i o n o f a n t i b o d i e s c o v a l e n t l y bound i n random o r i e n t a t i o n s can be a t t a c h e d v i a l i n k ­ ages c l o s e t o a combining s i t e , e f f e c t i v e l y b l o c k i n g i t . Second, antibody-antibody s t e r i c e f f e c t s could hinder the a b i l i t y o f an a n t i g e n t o b i n d t o a c o m b i n i n g s i t e , t h e r e b y r e d u c i n g t h e number o f "active" sites. T h i r d , lowered s p e c i f i c a c t i v i t y i s expected f o r antibody molecules immobilized i n regions o f the support e i t h e r p a r t i a l l y o r completely i n a c c e s s i b l e t oantigens. And f i n a l l y , a d i f f e r e n t type o f i n a c t i v a t i o n w i l l r e s u l t i f adverse conforma­ t i o n a l changes occur as a r e s u l t o f the i m m o b i l i z a t i o n p r o c e s s . The a i m o f t h i s r e s e a r c h , t h e n , i s t o b e t t e r u n d e r s t a n d w h i c h

Asenjo and Hong; Separation, Recovery, and Purification in Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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PURIFICATION IN BIOTECHNOLOGY

o f t h e s e m e c h a n i s m s i s o r a r e i m p o r t a n t i n f o r m u l a t i n g immunos o r b e n t s o f o p t i m a l a c t i v i t y and b i n d i n g s t r e n g t h . To do t h i s i t i s n e c e s s a r y t o d e t e r m i n e t h e a c t i v i t y and c o n f o r m a t i o n o f a n t i bodies immobilized to a support. Unfortunately, the supports o f t e n used ( e . g . a g a r o s e s ) i n t e r f e r e w i t h most d i r e c t p h y s i c a l measurements o f p r o t e i n s t r u c t u r e and f u n c t i o n . One m e t h o d l a r g e l y i n s e n s i t i v e t o t h e n a t u r e o f t h e s u p p o r t , h o w e v e r , i s EPR s p e c troscopy.

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M a t e r i a l s a n d M e t h o d s - EPR

Spectroscopy

EPR, o r e l e c t r o n p a r a m a g n e t i c r e s o n a n c e s p e c t r o s c o p y , i s a m a g n e t i c resonance technique designed to detect species c o n t a i n i n g unpaired electrons.(10-11) I t i s s i m i l a r i n p r i n c i p l e t o NMR, the primary d i f f e r e n c e b e i n g t h a t EPR i s b a s e d o n t h e m a g n e t i c moments o f u n p a i r e d e l e c t r o n s r a t h e r t h a n t h e m a g n e t i c moments o f s p e c i f i c nuclei. S i n c e a n t i b o d i e s d o n t n o r m a l l y p o s s e s s any u n p a i r e d electrons, small antigens, i . e . haptens, containing stable free r a d i c a l s ( " s p i n l a b e l s " ) c a n b e u s e d a l o n g w i t h EPR t o p r o b e t h e antibody combining s i t e . B y u t i l i z i n g t h e two d i f f e r e n t 2,4d i n i t r o p h e n y l (DNP) s p i n l a b e l s s h o w n i n F i g u r e 2, we a r e i n v e s t i g a t i n g t h e c o n f o r m a t i o n and a c t i v i t y o f a n t i - D N P m o n o c l o n a l a n t i b o d i e s i n s o l u t i o n and i m m o b i l i z e d by d i f f e r e n t methods. EPR s p e c t r o s c o p y i s a p o w e r f u l method f o r t h i s k i n d o f s t u d y because t h e EPR s p e c t r u m c a n p r o v i d e d e t a i l a b o u t t h e m o t i o n o f t h e s p i n l a b e l and t h u s t h e c o n f o r m a t i o n o f t h e a n t i b o d y c o m b i n i n g s i t e . In this w a y , EPR c a n h e l p t o e l u c i d a t e w h i c h o f t h e p o s s i b l e i n a c t i v a t i o n mechanisms d i s c u s s e d above i s o r are i m p o r t a n t . T

Shown i n F i g u r e 3 a r e EPR s p e c t r a o f s p i n l a b e l FDNP-SL i n s o l u t i o n a n d b o u n d t o a n t i - D N P a n t i b o d y . The m o r e r e s t r i c t e d m o t i o n o f a n t i b o d y - b o u n d a n t i g e n r e s u l t s i n b r o a d e r l i n e s h a p e s and a l a r g e r maximum p e a k - t o - p e a k s p l i t t i n g , t e r m e d & » The t h r e e peaked spectrum of s p i n l a b e l i n s o l u t i o n , f o r i n s t a n c e , i s c h a r a c t e r i z e d by an A o f a b o u t 34G, w h e r e a s t h e s p e c t r u m o f a n t i b o d y max b o u n d FDNP-SL h a s a n A o f 61G. I t s h o u l d a l s o be n o t e d t h a t t h e max a n t i b o d y bound s p e c t r u m i s a c o m p o s i t e spectrum c o n t a i n i n g b o t h t h e c h a r a c t e r i s t i c t h r e e - p e a k e d f r e e s p e c t r u m and a bound s p i n l a b e l spectrum of broader lineshape. S i n c e the s i g n a l i n t e n s i t y of each spectrum i s p r o p o r t i o n a l to the concentration of s p i n l a b e l , the h e i g h t s o f i s o l a t e d p e a k s a n d / o r t h e d o u b l e i n t e g r a l o f an e n t i r e a b s o r p t i o n s p e c t r u m c a n be u s e d t o d e t e r m i n e c o n c e n t r a t i o n s o f f r e e and bound s p i n l a b e l . ( 1 2 ) For i n s t a n c e , i n the case of the f r e e l a b e l , e i t h e r t h e p e a k h e i g h t o r d o u b l e i n t e g r a l c a n be u s e d . In the case of the composite spectrum, however, the i n t e g r a l p r o v i d e s t h e c o n c e n t r a t i o n o f b o t h f r e e and bound l a b e l , and i t i s n e c e s s a r y to s u b t r a c t the c o n c e n t r a t i o n of f r e e l a b e l from the t o t a l to d e t e r m i n e t h e a m o u n t o f s p i n l a b e l b o u n d t o a n t i b o d y . By d o i n g s o f o r samples c o n t a i n i n g d i f f e r e n t r a t i o s o f f r e e t o bound s p i n l a b e l , t h e e q u i l i b r i u m c o n s t a n t b e t w e e n a n t i b o d y and h a p t e n c a n be o b t a i n e d d i r e c t l y f r o m EPR s p e c t r a . A more s o p h i s t i c a t e d a n a l y s i s o f s p i n l a b e l m o t i o n i n t h e c o m b i n i n g s i t e i n v o l v e s t h e a n a l y s i s o f e x p e r i m e n t a l EPR s p e c t r a w i t h t h e a i d o f c o m p u t e r s i m u l a t i o n s , s u c h a s t h o s e o f F r e e d et at. m a K

Asenjo and Hong; Separation, Recovery, and Purification in Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

15.

EPR Spectroscopy of Monoclonal Antibodies

F E R N A N D E Z ET AL.

-


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R e s u l t s and D i s c u s s i o n The b e s t f i t t o d a t e b e t w e e n s i m u l a t e d a n d e x p e r i m e n t a l s p e c t r a was o b t a i n e d w i t h s o l u b l e IgG^, w i t h β e q u a l t o 5 0 ° , τ = 5.5 n s , a n d χ

and

τ

ζ

= 33 n s .

This

represents

a fast

r o t a t i o n of the spin

1

l a b e l around t h e x a x i s and slower m o t i o n around t h e o t h e r axes as d e p i c t e d i n F i g u r e 4 b . I t i s i n t e r e s t i n g t o n o t e t h a t 33 n s a g r e e s w e l l w i t h t h e r o t a t i o n a l c o r r e l a t i o n t i m e o f 47 n s f o r t h e F , f r a g ab m e n t o f a n I g E m o l e c u l e d e t e r m i n e d b y S l a t t e r y , et al.(15) using steady s t a t e a n i s o t r o p y measurements. T h i s agreement suggests t h a t τ and τ r e p r e s e n t time c o n s t a n t s f o r r o t a t i o n a l m o t i o n o f t h e F , y ζ ab fragment as a whole. U n f o r t u n a t e l y , because o f t h e complex and t i m e - c o n s u m i n g n a t u r e o f t h e s i m u l a t i o n s , n o t h i n g f u r t h e r c a n be r e p o r t e d a t t h i s time about t h e motion o f t h e s p i n l a b e l i n t h e combining s i t e t h r o u g h c o r r e l a t i o n times and t i l t a n g l e s . However, t h e r e i s a l s o u s e f u l i n f o r m a t i o n c o n t a i n e d i n maximum p e a k - t o - p e a k splittings. In s t u d i e s t o date, d i f f e r e n t s p i n l a b e l s have produced d i f ­ f e r e n t s p e c t r a when c o m b i n e d w i t h d i f f e r e n t a n t i b o d i e s . F o r exam­ ple, t h e s p l i t t i n g s and b i n d i n g c o n s t a n t s f o r t h e two d i f f e r e n t s p i n l a b e l s b i n d i n g t o s o l u b l e a n t i - D N P IgG«, a n t i b o d i e s a r e zb s u m m a r i z e d i n T a b l e I . T h e s e d a t a s h o w t h a t EPR i s s e n s i t i v e t o d i f f e r e n c e s i n t h e m o t i o n o f ( 1 ) t h e same s p i n l a b e l i n d i f f e r e n t motional environments ( i . e . , d i f f e r e n t antibody combining s i t e s ) ,

Table I .

Maximum p e a k - t o - p e a k s p l i t t i n g s a n d e q u i l i b r i u m b i n d i n g c o n s t a n t s f o r s o l u b l e I g G , a n d I g E c o m b i n e d w i t h DNP-SL and FDNP-SL. A (gauss) max Κ (M ) Spin Labeled Hapten ?

Antibody

1

DNP-SL

2.2 χ Ι Ο

6

G

62

3.9 χ Ι Ο

6

G

FDNP-SL

56

1.3 χ Ι Ο 4.8 χ 1 0

7

^ 2b ^ 2b IgE IgE

DNP-SL FDNP-SL

61

6

I

Asenjo and Hong; Separation, Recovery, and Purification in Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

5 6

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15.

F E R N A N D E Z ET AL.

Figure

3.

EPR Spectroscopy of Monoclonal Antibodies

E P R s p e c t r a o f (A)

f r e e and

(B)

bound s p i n

213

label.

F i g u r e 4. S c h e m a t i c o f s p i n l a b e l i n a n t i b o d y c o m b i n i n g s i t e : (A) w i t h o u t " d i f f u s i o n a l t i l t " , ( B ) w i t h " d i f f u s i o n a l tilt".

Asenjo and Hong; Separation, Recovery, and Purification in Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

214

SEPARATION, RECOVERY, AND

PURIFICATION IN BIOTECHNOLOGY

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a n d o f ( 2 ) d i f f e r e n t s p i n l a b e l s i n t h e same e n v i r o n m e n t ( i . e . , t h e same a n t i b o d y c o m b i n i n g s i t e ) . T h i s s u g g e s t s t h a t EPR m i g h t b e a b l e t o d i s c r i m i n a t e m o t i o n a l d i f f e r e n c e s caused by b i n d i n g s i t e c o n f o r m a t i o n a l changes. I f t h i s i s t h e c a s e , EPR s h o u l d be a u s e ­ f u l tool i n studying structure-function relationships i n immobilized a n t i b o d i e s as w e l l . F o r i m m o b i l i z a t i o n s t u d i e s t o d a t e , two d i s t i n c t modes o f i m m o b i l i z a t i o n have been used. The f i r s t u t i l i z e s n o n s p e c i f i c c o v a l e n t b o n d i n g t o C N B r - a c t i v a t e d Sepharose v i a p r i m a r y amino g r o u p s on t h e a n t i b o d y m o l e c u l e . S i n c e t h e r e a r e many o f t h e s e a v a i l a b l e on t h e a n t i b o d y , t h i s i s e x p e c t e d t o r e s u l t i n random o r i e n t a t i o n o f a n t i b o d y m o l e c u l e s on t h e s u p p o r t . The o t h e r m e t h o d i n v o l v e s l i n k a g e t h r o u g h i m m o b i l i z e d p r o t e i n A, a p r o t e i n w h i c h binds immunoglobulins i n t h e s t r u c t u r a l F^ p o r t i o n o f t h e m o l e c u l e . (15) Because t h i s method s h o u l d r e s u l t i n i m m o b i l i z e d a n t i b o d y m o l e c u l e s w i t h more o p t i m a l o r i e n t a t i o n s , i t i s e x p e c t e d t o p r o d u c e s a m p l e s w i t h h i g h e r a c t i v i t y , a l t h o u g h i t w o u l d c e r t a i n l y be a m o r e e x p e n s i v e and c o m p l e x t e c h n i q u e t o c a r r y o u t on a l a r g e s c a l e . Shown i n T a b l e I I a r e a c t i v i t y a n d b i n d i n g c o n s t a n t d a t a f o r s a m p l e s o f i m m o b i l i z e d a n t i b o d i e s p r e p a r e d b y t h e two d i f f e r e n t methods. The l o a d i n g o f I g G o n t h e s u p p o r t was d e t e r m i n e d b y a m i n o a c i d a n a l y s i s , a n d t h e a m o u n t o f a c t i v e a n d a c c e s s i b l e a n t i b o d y was d e t e r m i n e d b y EPR s p e c t r o s c o p y . The b i n d i n g c o n s t a n t f o r t h e C N B r - S e p h a r o s e s a m p l e was d e t e r m i n e d b y f l u o r e s c e n c e t i t r a t i o n . ( 1 6 ) Both samples have lowered s p e c i f i c a c t i v i t y w i t h r e s p e c t to the i d e a l v a l u e o f 2.0 ( r e m e m b e r t h a t t h e r e a r e two c o m b i n i n g s i t e s p e r a n t i b o d y m o l e c u l e ) , and t h e b i n d i n g c o n s t a n t f o r t h e C N B r - S e p h a r o s e sample i s o n l y 44% t h a t o f i t s v a l u e i n s o l u t i o n . I t i s also very i n t e r e s t i n g to note that c o n t r a r y to expectations the P r o t e i n - A s a m p l e h a s a l o w e r s p e c i f i c a c t i v i t y , w h i c h may be d u e t o t h e f a c t t h a t i t h a s a much h i g h e r l o a d i n g t h a n t h e C N B r - S e p h a r o s e s a m p l e . C u r r e n t r e s e a r c h i n t h i s l a b o r a t o r y s h o u l d soon p r o v i d e a more d e f i n i t i v e explanation f o r these r e s u l t s .

Table

II.

L o a d i n g s , s p e c i f i c a c t i v i t i e s , and b i n d i n g c o n s t a n t s f o r s a m p l e s o f i m m o b i l i z e d IgG_, . Zb C o u p l e d IgG Active/Accessible by Amino A c i d I g G b y EPR Specific Κ Support A n a l y s i s (mg/ml) (mg/ml) Activity (1/M)

CNBr-Sepharose

0.58

0.35

1.2

1.7xl0 (44%)

ProteinASepharose

1.7

0.70

0.8

?

Asenjo and Hong; Separation, Recovery, and Purification in Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

6

15.

F E R N A N D E Z ET AL.

IgG^^

Shown i n F i g u r e 5 a r e E P R s p e c t r a o f FDNP-SL c o m b i n e d w i t h i n s o l u t i o n and i m m o b i l i z e d t o C N B r - S e p h a r o s e . Although an

accurate A cannot be determined from these p a r t i c u l a r s p e c t r a , max the l e f t - m o s t peak i n the spectrum of i m m o b i l i z e d a n t i b o d y i s s h i f t e d s i g n i f i c a n t l y w i t h respect t o i t s p o s i t i o n i n the spectrum of antibody i n s o l u t i o n . This s h i f t i n d i c a t e s that the o v e r a l l m o b i l i t y o f t h e s p i n l a b e l i s more r e s t r i c t e d when t h e l a b e l o c c u p i e s the combining s i t e o f i m m o b i l i z e d a n t i b o d y . The accompanying decrease i n the b i n d i n g c o n s t a n t observed upon i m m o b i l i z a t i o n i n d i c a t e s t h a t such changes i n s p i n l a b e l m o b i l i t y are due a t l e a s t i n p a r t t o changes i n combining s i t e c o n f o r m a t i o n ; h o w e v e r , a l t e r e d m o t i o n o f t h e e n t i r e F , f r a g m e n t may a l s o h a v e ab i n f l u e n c e d the spectrum. C l a r i f i c a t i o n o f t h i s p o i n t , provided by the use o f computer s i m u l a t i o n s t odetermine r o t a t i o n a l c o r r e l a t i o n times f o r the immobilized antibody system i s expected i n the near future. r

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215

EPR Spectroscopy of Monoclonal Antibodies

Concluding

r

Remarks

The d a t a p r e s e n t e d a b o v e e s t a b l i s h t h e a p p l i c a b i l i t y o f E P R t o s t r u c t u r e - f u n c t i o n c h a r a c t e r i z a t i o n o f i m m o b i l i z e d a n t i b o d i e s . The soluble A d e t e r m i n a t i o n s i n d i c a t e t h a t EPR i s s e n s i t i v e t o max d i f f e r e n t e n v i r o n m e n t s and t o t h e s t r u c t u r e o f t h e p r o b e u s e d . They a l s o s u g g e s t t h a t d i f f e r e n t p r o b e s m i g h t b e used t o o b t a i n a more c o m p l e t e p i c t u r e o f t h e c o n f o r m a t i o n o f t h e b i n d i n g s i t e . I n a d d i t i o n , t h e m e a s u r a b l e d i f f e r e n c e s between t h e EPR s p e c t r a o f s o l u b l e and i m m o b i l i z e d a n t i b o d i e s i n d i c a t e t h a t i m m o b i l i z a t i o n has s i g n i f i c a n t l y a f f e c t e d the motion o f the s p i n l a b e l i n the combining s i t e . A l t h o u g h the d a t a p r e s e n t e d h e r e do not a l l o w s p e c i f i c c o n c l u s i o n s t obe drawn about the e f f e c t o f i m m o b i l i z a t i o n on a n t i b o d y c o n f o r m a t i o n and a c t i v i t y , t h e y do i l l u s t r a t e t h a t EPR can p r o v i d e m o l e c u l a r l e v e l i n s i g h t s not a v a i l a b l e by t r a d i t i o n a l methods. Thus, EPR s p e c t r o s c o p y s h o u l d s e r v e a s a p o w e r f u l t o o l

F i g u r e 5. IgG .

EPR s p e c t r a o f s o l u b l e and

CNBr

Sepharose-immobilized

2 b

Asenjo and Hong; Separation, Recovery, and Purification in Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

216

S E P A R A T I O N , R E C O V E R Y , A N D P U R I F I C A T I O N IN B I O T E C H N O L O G Y

for d i r e c t experimental analyses o f immobilized antibody structure and f u n c t i o n , a n d t h e r e f o r e h e l p t o e l u c i d a t e t h e b a s i s f o r t h e s u b o p t i m a l b i n d i n g c a p a c i t i e s f r e q u e n t l y e x h i b i t e d b y immuno­ sorbents . Acknowledgments The a u t h o r s a r e i n d e b t e d t o D r . B a r b a r a B a i r d , D. D a v i d H o l o w k a , and D r . N o r m a l K l i n m a n f o r t h e i r a s s i s t a n c e i n o b t a i n i n g a n t i b o d i e s and t o Dave S c h n e i d e r a n d G l e n n M i l l h a u s e r f o r t h e i r a d v i c e o n t h e spectral simulations. T h i s m a t e r i a l i s based upon work supported under a N a t i o n a l Science Foundation Graduate F e l l o w s h i p .

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Literature Cited 1. Turkova, J. In "Affinity Chromatography"; Elsevier: New York, 1975; p. 95. 2. Chase, H. A. Chem. Eng. Sci. 1984, 39, 1099. 3. Goding, J. W. In "Monoclonal Antibodies: Principles and Practice"; Academic: New York, 1983; pp. 188-207. 4. Mukkur, T. K. S. Biochem. J. 1978, 172, 39. 5. Dwyer, J. W. Bio/Technology 1984, 2, 957. 6. Low, D. "Proceedings of Biotech 83, International Conference on the Commercial Applications and Implications of Biotech­ nology"; Online: Northwood, U.K. 1983, p. 830. 7. Bolton, A. E.; Hunter, W. M. Biochim. Biophys. Acta 1973, 329, 318. 8. Eveleigh, J. W.; Levy, D. E. J. Solid-Phase Biochem. 1977, 2, 45. 9. Weston, P. D.; Scorer, R. In "Affinity Chromatography"; Hoffman-Ostenhof, et al., Ed.; Pergamon: New York, 1978, p. 207. 10. Schumacher, R. T. "Magnetic Resonance"; W. A. Benjamin: New York, 1970. 11. Cantor, C. R.; Schimmel, P. R. In "Biophysical Chemistry"; W. H. Freeman: New York, 1980; pp. 525-536. 12. Bailey, J. E.; Clark, D. S. In "Methods in Enzymology"; Mosbach, K., Ed.; Academic: New York, in press. 13. Meirovitch, E.; Igner, D.; Igner, E.; Moro, G.; Freed, J. H. J. Chem. Phys. 1982, 77, 3915. 14. Goldman, S. Α.; Bruno, G. V.; Polnaszek, C. F.; Freed, J. H.; J. Chem. Phys. 1972, 56, 716; Hwang, J. S.; Mason, R. P.; Hwang, L. P.; Freed, J. H. J. Phys. Chem. 1975, 79, 289; J. Phys. Chem. 1975, 79, 2283; Campbell, R. F.; Freed, J. H. J. Phys. Chem. 1980, 84, 2668; Meirovitch, E.; Freed, J. H. J. Phys. Chem. 1980, 84, 2459; Shiotani, M.; Moro, G.; Freed, J. H. J. Chem. Phys. 1981, 74, 15. 15. Slattery, J.; Holowka, D. A.; Baird, B. A. Biochemistry, in press. 16. Goding, J. W. In "Monoclonal Antibodies: Principles and Practice"; Academic: New York, 1983; p. 195. 17. Mukkur, T. K. S.; Szewczuk, M. R.; Schmidt, Ε. E., Jr. Immunochem. 1974, 11, 9. Received April 16, 1986

Asenjo and Hong; Separation, Recovery, and Purification in Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1986.