Chapter 13
The Impact of Chemistry on Biotechnology Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SANTA BARBARA on 03/02/18. For personal use only.
Analytical Challenges in Biotechnology John B. Landis Upjohn Company, Kalamazoo, MI 49001
The development of r-DNA and hybridoma technology has revolutionized the application of biotechnology for the production of peptides and proteins in the laboratory and on a commercial scale. Using this new technology, it is now possible to produce biopolymers in large quantities with high purity. In contrast, proteins and peptides have historically been produced through isolation from natural sources as heterogeneous mixtures of low purity. There are important differences between the small organic molecules typically encountered by the analytical chemist and the proteins and peptides produced from biotechnology. While most typical organic molecules have molecular weights less than 1,000 Daltons, the products of biotechnology will typically have molecular weights between 1,000 - 1,000,000 Dal tons. Unlike their small molecule counterparts, the three dimensional structure of proteins and peptides dictates function and activity and is not entirely controlled by the chemical structure of the molecule. The larger molecules are likely to be isolated from a complex matrix in which they are a minor constituent among many species of similar size and composition. To meet these challenges, new analytical strategies and methods are under development for the characterization and analysis of biopolymers. The routine application of cloning and expression of a large number of proteins will challenge the analytical chemist to rapidly characterize and analyze these molecules. Important parameters to characterize and control are identity (structure), purity, and impurity. An understanding of protein structure is important to guide the design of new proteins, substrates, and inhibitors; to enhance protein stability and handling characteristics; and to modify biological function. A knowledge of protein structure is an essential ingredient in the development of a scheme for the batch-to-batch control of protein identity. Protein identity is determined by measuring composition, sequence, and conformation. The developm
0097-6156/88/0362-0174$06.00/0 © 1988 American Chemical Society
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Analytical Challenges in Biotechnology
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The Impact of Chemistry on Biotechnology Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SANTA BARBARA on 03/02/18. For personal use only.
o f m i c r o c h e m i c a l methods f o r the d e t e r m i n a t i o n o f amino a c i d compos i t i o n and sequence has g r e a t l y improved the s p e e d , a c c u r a c y , and precision. A d d i t i o n a l improvements a r e needed i n s e n s i t i v i t y , r e l i a b i l i t y , and data a n a l y s i s . Advances i n s p e c t r o s c o p y have i n c r e a s e d our d e t a i l e d knowledge o f p r o t e i n c o n f o r m a t i o n . S p e c t a c u l a r advances have been made i n the development o f h i g h r e s o l u t i o n t e c h n i q u e s such as x - r a y , NMR, and Raman s p e c t r o s c o p y . X - r a y c r y s t a l l o g r a p h y has p r o v i d e d the most d e t a i l e d and comprehens i v e p i c t u r e o f t h r e e - d i m e n s i o n a l p r o t e i n s t r u c t u r e i n the s o l i d state. The advent o f h i g h i n t e n s i t y s o u r c e s and new d e t e c t o r s promises to reduce the time r e q u i r e d to o b t a i n a s t r u c t u r e and to improve the a c c u r a c y o f the s t r u c t u r e . Recent p r o g r e s s i n NMR i s e x t e n d i n g the d e t e r m i n a t i o n o f d e t a i l e d s t r u c t u r e on small p r o t e i n s in solution. Many s t r u c t u r a l problems a r e a c c e s s i b l e o n l y through NMR, n o t a b l y where c r y s t a l l i n e m a t e r i a l s cannot be o b t a i n e d . The advent o f the t u n a b l e dye l a s e r , h i g h powered p u l s e d l a s e r s , and the e x t e n s i o n o f Raman to the UY has g r e a t l y i n c r e a s e d the s e n s i t i v i t y and s e l e c t i v i t y o f t h i s t e c h n i q u e f o r examining the p e p t i d e backbone and s u l f h y d r y l g r o u p s . Major advances i n Mass S p e c t r o s c o p y has extended t h i s t e c h nique to the sequence o f p e p t i d e s , DNA, and c a r b o h y d r a t e s . Optical methods such as a b s o r b a n c e , f l u o r e s c e n c e , and c i r c u l a r d i c h r o i s m a r e u s e f u l t e c h n i q u e s f o r f o l l o w i n g s t r u c t u r a l changes i n s o l u tion. Recent work has i n d i c a t e d t h a t monoclonal a n t i b o d i e s r a i s e d a g a i n s t c o n f o r m a t i o n a l ^ i m p o r t a n t e p i t o p e s may a l s o s e r v e as a conformational i d e n t i t y t e s t . The d e t e r m i n a t i o n o f p r o t e i n p u r i t y and i m p u r i t y a r e i m p o r t a n t f o r the e s t a b l i s h m e n t o f p r o d u c t s a f e t y and e f f e c t i v e n e s s . Since r-DNA produced p r o t e i n s a r e o f t e n i s o l a t e d as a minor component from a f e r m e n t a t i o n p r o c e s s , p o t e n t i a l i m p u r i t i e s might i n c l u d e h o s t c e l l p r o t e i n s , r e s i d u a l DNA, p r o d u c t - r e l a t e d i m p u r i t i e s i n c l u d i n g d e g r a d a t i o n p r o d u c t s , p r o c e s s r e a g e n t s and b i o l o g i c a l c o n t a m i n a n t s such as v i r u s e s , e n d o t o x i n , p y r o g e n s , and b a c t e r i a . The p r e s e n c e o f many o f t h e s e i m p u r i t i e s i n t r a c e amounts may have a n e g a t i v e impact on p r o d u c t q u a l i t y . Protein contaminants, f o r example, may cause an immune r e s p o n s e a t the ppm l e v e l when used as a drug. The d e t e c t i o n and a c c u r a t e q u a n t i t a t i o n o f c o n t a m i n a t i n g p r o t e i n s d i f f e r i n g by as l i t t l e as one amino a c i d i n a complex m a t r i x i s an a n a l y t i c a l c h a l l e n g e t h a t n e c e s s i t a t e s the development o f new approaches to a n a l y s i s . C u r r e n t l y , immunochemical methods a r e the o n l y t e c h n i q u e s t h a t approach t h i s s e n s i t i v i t y and s e l e c tivity. The use o f monoclonal a n t i b o d i e s w i l l make t h e s e methods more r e l i a b l e . Contaminants t h a t may a l s o a r i s e from a l t e r a t i o n s i n m o l e c u l a r s t r u c t u r e d u r i n g the p r o d u c t i o n p r o c e s s i n c l u d e h e t e r o g e n e i t y i n amino a c i d s e q u e n c e , chemical and p h y s i c a l a l t e r a t i o n v i a i s o l a t i o n s and p u r i f i c a t i o n , and p o s t t r a n s i a t i o n a l m o d i f i c a t i o n s v i a mechanisms such as g l y c o s y l a t i o n . A n a l y t i c a l methods and i n s t r u m e n t a t i o n a r e needed t o meet the c h a l l e n g e f o r the c o m m e r c i a l i z a t i o n o f b i o t e c h n o l o g y . P r o t e i n s can now be produced much f a s t e r than they can be c h a r a c t e r i z e d . New a n a l y t i c a l schemes are needed to c h a r a c t e r i z e p r o t e i n s as c h e m i cals. The added c o m p l e x i t y o f p r o t e i n s make i t i m p o s s i b l e t o
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T H E IMPACT OF CHEMISTRY ON BIOTECHNOLOGY
The Impact of Chemistry on Biotechnology Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SANTA BARBARA on 03/02/18. For personal use only.
c h a r a c t e r i z e them as f u l l y as the small m o l e c u l e s t y p i c a l l y e n c o u n t e r e d by the a n a l y t i c a l c h e m i s t . Many o f the methods c u r r e n t l y b e i n g used f o r p r o t e i n c h a r a c t e r i z a t i o n and a n a l y s i s were d e v e l o p e d f o r p r o t e i n s as b i o l o g i c a l s and not f o r p r o t e i n s as c h e m i c a l s . A new g e n e r a t i o n o f methods and s t r a t e g i e s a r e needed to i n c r e a s e s e n s i t i v i t y , s e l e c t i v i t y , p r e c i s i o n , and a c c u r a c y . RECEIVED September 3, 1987
