Protein Purification - ACS Publications - American Chemical Society

Chapter 2

Strategies for Large-Scale Protein Purification

Sa V. Ho

Downloaded by GEORGETOWN UNIV on December 5, 2015 | http://pubs.acs.org Publication Date: June 12, 1990 | doi: 10.1021/bk-1990-0427.ch002

Bio-Products Division, Eastman Kodak Company, Rochester,NY14652-3605

The development of economical processes for purifying proteins from recombinant sources requires the integration of many isolation and purification methods as well as healthy cross-exchange among molecular biology, fermentation, purification and analytical groups. With regards to purification, two schools of thought seem to have emerged. One approach is to start with a highly specific method early on to achieve the required purification in a single step. While aesthetically appealing, in practice this approach lacks a truly high resolution method that is also economical and scalable. The second approach utilizes a cascade of conventional methods to achieve the required protein purity. Here, what methods to use and in what order represent a major task in the process development effort. Based on the author's industrial experience and process information from the literature, some general guidelines for developing optimal purification processes could be established. Examples showing the applicability of this approach will be discussed. The development o f p u r i f i c a t i o n p r o c e s s e s f o r l a r g e - s c a l e manufacture o f p r o t e i n s i s a v e r y c h a l l e n g i n g a c t i v i t y . While p r o t e i n p u r i f i c a t i o n i t s e l f i s a l r e a d y complex, the r e q u i r e m e n t o f " l a r g e s c a l e " imposes a d d i t i o n a l i m p l i c a t i o n s such as economy, s c a l a b i l i t y , and r e p r o d u c i b i l i t y , which s e v e r e l y c o n s t r a i n what can and have t o be done. The f o c u s i n t h i s paper i s not on o p t i m i z i n g ( o r a d v o c a t i n g ) any p a r t i c u l a r p u r i f i c a t i o n method, f o r which one c o u l d c o n s u l t e x p e r t s i n t h e f i e l d o r draw on the w e a l t h o f literature available. R a t h e r , we w i l l t r y t o t a c k l e the c h a l l e n g i n g t a s k o f how t o c o n v e r t a f e r m e n t a t i o n b r o t h o r crude s o l u t i o n i n t o the p u r i f i e d p r o d u c t t h a t s a t i s f i e s a l l the r e q u i r e m e n t s ( c o s t , purity, efficacy, etc.). Only r e c e n t l y have s e v e r a l e x c e l l e n t p u b l i c a t i o n s appeared a d d r e s s i n g v a r i o u s a s p e c t s o f p r o c e s s development f o r l a r g e - s c a l e p r o t e i n p u r i f i c a t i o n ( 1 - 3 ) .

0097-6156/90/0427-0014$06.25/0 © 1990 American Chemical Society

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

2. HO


15

STRATEGY DISCUSSION


P u r i f i c a t i o n p r o c e s s development, e s p e c i a l l y f o r r D N A - d e r i v e d products, i s a m u l t i f a c e t e d a c t i v i t y that requires close p a r t i c i p a t i o n o f many s c i e n t i f i c d i s c i p l i n e s as i l l u s t r a t e d i n Figure 1. The s i g n i f i c a n c e and the i n t e r - r e l a t i o n s h i p o f these elements a r e the f o c u s o f t h i s d i s c u s s i o n . PURIFICATION PROCESS. U n l i k e f e r m e n t a t i o n development which b a s i c a l l y i n v o l v e s o p t i m i z i n g the o p e r a t i n g p a r a m e t e r s i n a more o r l e s s s t a n d a r d v e s s e l , t h e r e i s not a s i n g l e t e c h n i q u e o r equipment t h a t i s c a p a b l e o f d e l i v e r i n g p u r i f i e d p r o t e i n i n i t s f i n a l form d i r e c t l y from the f e r m e n t a t i o n b r o t h . So the f i r s t major problem c o n f r o n t i n g a p u r i f i c a t i o n development p e r s o n i s , among the f a i r l y l a r g e number o f t e c h n i q u e s and equipment a v a i l a b l e , n o t o n l y what t e c h n i q u e s s h o u l d be used, but a l s o i n what o r d e r (sequence) as w e l l as how each one i s o p e r a t e d . These t h r e e a s p e c t s a r e i n t e r r e l a t e d and a r e d e t e r m i n e d by the f o l l o w i n g two main f a c t o r s . The f i r s t one i s the n a t u r e o f the s t a r t i n g s o l u t i o n , which can come from many d i f f e r e n t s o u r c e s ( m i c r o b e s , t i s s u e c u l t u r e , s y n t h e s i s ) , w i t h the p r o d u c t i n d i f f e r e n t forms ( s o l u b l e o r as i n c l u s i o n b o d i e s ) and at d i f f e r e n t l o c a t i o n s ( c y t o p l a s m , p e r i p l a s m , i n the b r o t h ) . Each s i t u a t i o n imposes d i f f e r e n t c o n s t r a i n t s and c h a l l e n g e s t o the development e f f o r t . The second key f a c t o r r e l a t e s to the p r o d u c t . A s p e c t s such as p u r i t y , form, i m p u r i t y p r o f i l e , e t c . ( p r o d u c t s p e c i f i c a t i o n ) and c o s t s t r o n g l y d i c t a t e what an a c c e p t a b l e p u r i f i c a t i o n p r o c e s s would be l i k e . F o r both c o s t and p u r i t y r e q u i r e m e n t the spectrum spans from human t h e r a p e u t i c s such as t-PA, i n s u l i n , hGH ( h i g h c o s t , u l t r a p u r i t y ) t o a n i m a l growth hormones (medium c o s t and h i g h p u r i t y ) to i n d u s t r i a l enzymes where the c o s t i s low but p u r i t y i s not so critical. THE ROLE OF FERMENTATION. F e r m e n t a t i o n c o n d i t i o n s and p r o t o c o l s determine the q u a n t i t y as w e l l as the q u a l i t y o f the s t a r t i n g material (concentration, conformation, p u r i t y , impurity p r o f i l e ) , which g r e a t l y impacts downstream p r o c e s s i n g . The o p t i m i z a t i o n ( o p e r a t i n g c o n d i t i o n s , raw m a t e r i a l s used, e t c . ) , t h e r e f o r e , s h o u l d not be based s o l e l y on performance a t the f e r m e n t a t i o n s t a g e . Due to the complex n a t u r e o f the b r o t h , h i g h e r t i t e r measured by HPLC o r SDS-PAGE, f o r example, may not be d i r e c t l y r e l a t e d t o the f i n a l amount o f p u r i f i e d , a c t i v e p r o d u c t o b t a i n e d . A peak on the HPLC o r a band on the g e l may c o n t a i n more than one component, a r i s i n g from s m a l l d i f f e r e n c e s i n amino a c i d r e s i d u e s o r d i f f e r e n t conformations. A l e s s well-known impact o f f e r m e n t a t i o n on p u r i f i c a t i o n i s t h a t s u b t l e m o d i f i c a t i o n s o f the p r o d u c t s r e s u l t i n g i n a m i x t u r e o f c l o s e l y - r e l a t e d compounds t h a t can be e x t r e m e l y d i f f i c u l t t o s e p a r a t e may be r e s o l v e d a t the f e r m e n t a t i o n s t a g e . An e x c e l l e n t example o f t h i s i s the work done by Amgen s c i e n t i s t s on n o r l e u c i n e misincorporation i n i n t e r l e u k i n - 2 ( 6 , 7 ) · They found t h a t the i n c o r p o r a t i o n o f n o r l e u c i n e i n s t e a d o f m e t h i o n i n e i n the p r o d u c t , which r e s u l t s i n p r o d u c t h e t e r o g e n e i t y w i t h u n p r e d i c t a b l e immunogenic consequences, c o u l d be m i n i m i z e d o r even e l i m i n a t e d by


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

nature of p r o t e i n

ANALYTICAL

Process Economics

(conditions)

(sequence)

How

(techniques)

What

FORMULATION

purification

. endotoxins - Activity

. nucleic acids

. proteins

- Contaminants

- Purity

Specifications

PURIFIED PROTEIN

(Porath et al)

Where

Secretion

- Metal Chelation -

. Peptide S y n t h e s i s

. T i s s u e Culture

Engineering

- PolyArg tail (Searle) - Fusion

Figure 1. The m u l t i f a c e t e d p r o c e s s development.

extracellular

. insoluble

. soluble

intracellular

Microbial:

STARTING MIXTURE

FERMENTATION

Genetic


ο 2

1

a

3 2

3

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17


s i m p l y adding l e u c i n e and/or methionine f e r m e n t a t i o n medium.

a t low l e v e l s

to the


FORMULATION CONSTRAINTS. F o r m u l a t i o n c o u l d impose f u r t h e r c o n s t r a i n t s on p r o d u c t c h a r a c t e r i s t i c s s i n c e the p r o d u c t i n i t s d e s i r e d d e l i v e r a b l e form ( s t a b i l i t y , a c t i v i t y , r e l e a s e r a t e , c o l o r , e t c . ) i s t r u l y the f i n a l g o a l . I t i s possible that a p r e f e r r e d p u r i f i c a t i o n p r o c e s s p u r e l y from a p r o c e s s i n g s t a n d p o i n t may produce a l e s s d e s i r a b l e p r o d u c t from a f o r m u l a t i o n s t a n d p o i n t (such as s t a b i l i t y , impurity p r o f i l e , delivery rate). So a p u r i f i e d p r o d u c t s h o u l d be r a p i d l y c a r r i e d through f o r m u l a t i o n and t e s t i n g t o ensure that i t i s acceptable before a p u r i f i c a t i o n process i s locked i n . THE IMPACT OF GENETIC ENGINEERING. The advent o f g e n e t i c e n g i n e e r i n g has p r o b a b l y e x e r t e d the b i g g e s t impact on p u r i f i c a t i o n p r a c t i c e , i n t r o d u c i n g new c h a l l e n g e s as w e l l as o f f e r i n g new solutions. I t i s p o s s i b l e to not o n l y a c h i e v e h i g h p r o d u c t i o n o f a p r o t e i n p r o d u c t but a l s o modify i t f o r improved s e p a r a t i o n and/or s t a b i l i t y ( e . g . , p o l y a r g i n i n e t a i l ( 8 ) , metal a f f i n i t y s i t e , f u s i o n p r o t e i n s ) or f o r determining i t s eventual residence (cytoplasmic, periplasmic or e x t r a c e l l u l a r ) . In a d d i t i o n , f o r some p r o t e i n s the number o f c y s t e i n e r e s i d u e s c o u l d be changed f o r improved r e f o l d efficiency ( 9 ) . THE ROLE OF ANALYTICAL. While not o b v i o u s , a n a l y t i c a l p l a y s a v e r y c r i t i c a l r o l e i n p r o c e s s development f o r rDNA-derived p r o d u c t s . P r o v i d i n g a q u a n t i t a t i v e assay f o r the p r o d u c t i s o n l y a s m a l l p a r t o f a n a l y t i c a l development. The a b i l i t y to i d e n t i f y contaminants t h a t not o n l y a r e p r e s e n t a t v e r y low c o n c e n t r a t i o n s but a l s o d i f f e r v e r y s l i g h t l y from the main p r o d u c t ( e . g . due to m i s i n c o r p o r a t i o n , p r o c e s s - r e l a t e d chemical m o d i f i c a t i o n or aggregation) i s a b s o l u t e l y e s s e n t i a l i n the o v e r a l l e f f o r t o f d e v e l o p i n g an o p t i m a l p u r i f i c a t i o n process. In summary, the o p t i m i z a t i o n o f a p u r i f i c a t i o n p r o c e s s r e q u i r e s c l o s e i n t e r a c t i o n s among a l l o f the above f u n c t i o n s , c a r e f u l p l a n n i n g as w e l l as a l l o w i n g room f o r i t e r a t i o n s . This i s , however, an i d e a l s i t u a t i o n . In r e a l i t y , the p r e s s u r e o f e a r l y market i n t r o d u c t i o n and slow r e g u l a t o r y c l e a r a n c e u s u a l l y f o r c e s one to l o c k i n w i t h an " i n f e r i o r " p r o c e s s , which c o u l d be f r u s t r a t i n g but n e c e s s a r y . J u s t b e i n g aware o f a l l these i s s u e s i s i n i t s e l f an important element o f p r o c e s s development. Then e a r l y p l a n n i n g i n combination w i t h a sound s t r a t e g y based on e x p e r i e n c e i s p r o b a b l y the b e s t one can do under the c i r c u m s t a n c e s . STRATEGY DEVELOPMENT The q u e s t i o n here i s how t o c o n v e r t an impure p r o t e i n s o l u t i o n such as a f e r m e n t a t i o n b r o t h i n t o a p u r i f i e d p r o d u c t t h a t meets a l l the requirements. I s t h e r e a methodology t h a t would o f f e r some guidelines? There seems t o be two g e n e r a l approaches. In one, a h i g h l y s p e c i f i c method such as i m m u n o a f f i n i t y i s u t i l i z e d e a r l y i n the p r o c e s s to a c h i e v e the maximum degree o f p u r i f i c a t i o n ( e . g .


18

PROTEIN PURIFICATION


a l p h a - i n t e r f e r o n , Hoffmann La Roche). T h i s approach i s not g e n e r i c , c o u l d be c o s t l y , and, i f a n t i b o d i e s are used as l i g a n d s , may have o t h e r o p e r a t i n g c o m p l i c a t i o n s such as s i g n i f i c a n t l o s s o f b i n d i n g c a p a c i t y upon i m m o b i l i z a t i o n , and l e a c h i n g o f the a n t i b o d i e s i n t o the p r o d u c t stream. A n t i b o d i e s may a l s o b i n d to the d e n a t u r e d , u n f o l d e d , m i s p a i r e d , and a g g r e g a t e d forms o f the p r o d u c t and i t s analogs. The o t h e r approach i s to use a s e r i e s o f " t r a d i t i o n a l " methods i n a c o n c e r t e d way to a c h i e v e the d e s i r e d g o a l ( p u r i t y , y i e l d , cost, s c a l a b i l i t y ) . The l a t t e r approach appears to be q u i t e e f f e c t i v e and has been used c o m m e r c i a l l y f o r p u r i f y i n g i n s u l i n (E. L i l l y ) and human growth hormone (Genentech) and i s p r o b a b l y used i n development stage f o r o t h e r p r o t e i n p r o d u c t s such as f i b r o b l a s t i n t e r f e r o n , i n t e r l e u k i n - 1 and animal growth hormones. A c t u a l l y the d i v i s i o n between the two approaches can be arbitrary. S i n c e no s i n g l e t e c h n i q u e c o u l d d e l i v e r the f i n a l p u r i f i e d p r o d u c t d i r e c t l y from the f e r m e n t a t i o n b r o t h o r crude e x t r a c t , h i g h l y s p e c i f i c methods such as i m m u n o a f f i n i t y still r e q u i r e the s u p p o r t o f o t h e r methods to a c c o m p l i s h the t a s k , e s p e c i a l l y i n l i g h t o f some o f i t s "unexpected" drawbacks d i s c u s s e d above. Thus, i n e i t h e r case p r o c e s s o p t i m i z a t i o n i s s t i l l required. The key d i f f e r e n c e i n the two approaches i s i n the i n i t i a l focus. In g e n e r a l , u n l e s s a h i g h l y s p e c i f i c and unique a f f i n i t y method has a l r e a d y been i d e n t i f i e d , we recommend t h a t the approach d e v e l o p e d below be used. B o n n e r j e a e t a l ( 2 ) a n a l y z e d 1 0 0 p u b l i c a t i o n s on l a b - s c a l e p r o t e i n p u r i f i c a t i o n procedures. They p l o t t e d the r e s u l t s ( F i g u r e 2 ) showing the number o f s t e p s used i n the p u r i f i c a t i o n scheme as a f u n c t i o n o f the f r e q u e n c y a method i s used i n each s t e p . A pattern seems to emerge. Homogenization i s the most f r e q u e n t l y used f i r s t s t e p , p r o b a b l y because most p r o t e i n p r o d u c t s are i n t r a c e l l u l a r , hence the need to break the c e l l s open. I t i s i n t e r e s t i n g that p r e c i p i t a t i o n i s p o p u l a r as the f i r s t p u r i f i c a t i o n s t e p , f o l l o w e d by more r e s o l v i n g methods such as ion-exchange and a f f i n i t y methods, and f i n i s h e d w i t h g e l f i l t r a t i o n . While these e x a c t methods are c e r t a i n l y not g e n e r a l l y a p p l i c a b l e , they seem to suggest a f a i r l y s e n s i b l e sequence o f e v e n t s . Going beyond these s p e c i f i c t e c h n i q u e s and combining our e x p e r i e n c e w i t h o t h e r s i n the l i t e r a t u r e r e g a r d i n g p u r i f y i n g p r o t e i n s from recombinant s o u r c e s , we propose the g e n e r a l p u r i f i c a t i o n scheme shown i n F i g u r e 3 . These are b l o c k s o f a c t i v i t i e s t h a t may r e q u i r e more than one s t e p . The essence o f the proposed scheme i s the d e l i b e r a t e b r e a k i n g down o f the development a c t i v i t y f o r p r o t e i n p u r i f i c a t i o n i n t o two s e p a r a t e b l o c k s : g r o s s p u r i f i c a t i o n and h i g h - r e s o l u t i o n purification. In g r o s s p u r i f i c a t i o n , the f o c u s i s to u t i l i z e a simple y e t e f f e c t i v e s t e p ( s ) t o s i g n i f i c a n t l y c l e a n up the s o l u t i o n i n such a way t h a t i t f l o w s n a t u r a l l y i n t o the next b l o c k where h i g h r e s o l u t i o n methods are used f o r the f i n a l p u r i f i c a t i o n . T h i s way, i t i s the g r o s s p u r i f i c a t i o n t h a t d e t e r m i n e s what and how h i g h - r e s o l u t i o n methods s h o u l d be used. There are s e v e r a l advantages w i t h t h i s approach. For one, t h e r e always e x i s t a l a r g e number o f i m p u r i t i e s i n the crude s o l u t i o n t h a t have extreme p r o p e r t i e s ( h i g h l y charged, e i t h e r v e r y l a r g e o r v e r y s m a l l , h i g h l y h y d r o p h o b i c , e t c . ) and t h a t c o u l d be removed e a s i l y i n a simple s t e p i f the a p p r o p r i a t e method i s used. Only a f t e r t h i s treatment i s i t



HO


I

2

3

4

5

6

7

Stage in purification scheme Figure 2 . A n a l y s i s o f the p u r i f i c a t i o n methods used at s u c c e s s i v e s t e p s i n the p u r i f i c a t i o n schemes (Reproduced w i t h p e r m i s s i o n from Ref. 2 C o p y r i g h t 1 9 8 6 Nature P u b l i s h i n g Company.)


20


. Fermentation Synthesis

. Tissue Culture


. Peptide Synthesis

. Homogenization .

. Centrifugation

Isolation &

. Filtration

Concentration

. L i q / L i q Ext. Refold . Adsorption

Gross

. Precipitation

Purification

. L i q / L i q Ext.

Hi-resolution

Chromatography

Purification

Concentration (Buffer Ex.)

Affinity

.

Interactions

Ultrafiltration

. Gel Filtration

Sterile Filt. Drying

— Figure

3-

Proposed

ι

—

general

purification

scheme.


2. HO

21


c l e a r what contaminants are l e f t . These tend to have s i m i l a r p r o p e r t i e s to the p r o d u c t o f i n t e r e s t . F o c u s i n g on s e p a r a t i o n o f s i m i l a r compounds, f r e e from o t h e r i n t e r f e r i n g contaminants, will r e s u l t i n h i g h e r c a p a c i t y , b e t t e r r e s o l u t i o n and l o n g e r l i f e f o r the more complex and more c o s t l y h i g h r e s o l u t i o n s t e p . The above scheme a u t o m a t i c a l l y o r g a n i z e s the l a r g e number o f i s o l a t i o n / p u r i f i c a t i o n t e c h n i q u e s i n t o a few manageable c a t e g o r i e s as shown i n F i g u r e 3 a l o n g w i t h the flow diagram and expanded i n T a b l e s I-IV. These b l o c k a c t i v i t i e s are c o n s i d e r e d i n d e t a i l below.


Table a)

b)

Cell

I.

Isolation

Rupture ο Homogenization ο E x t r a c t i o n (Bio/Chemical

S o l i d s / L i q u i d Separation ο Centrifugation ο Filtration ο Cross-Flow UF/MF ο Aqueous Two-Phase

Table

Methods

Treatment)

Partitioning

II.

Refold

a)

Dissolution ο Dénaturants ( u r e a , guanidine.HC1, SDS, e t c . ) and/or extreme pH

b)

Oxidation ο Complex, y i e l d l o s s due to a g g r e g r a t i o n ο Key parameters: pH, T, time con. o f dénaturants additives ο Genetic Engineering: 3 S-H • 2 S-H ( b e t a - i n t e r f e r o n )

Table

III.

Bulk

charged,

Purification

ο

Adsorption:

hydrophobic,

ο

Precipitation - pH - Temperature - Salts - Polymers (Neutral/Charged) - Organic S o l v e n t s * Affinity?

ο

Liquid/Liquid Extraction - Organic/Aqueous - Aqueous/Aqueous

Methods

affinity

adsorbents


22


Table


ο

IV.

High

R e s o l u t i o n Methods

Chromatography - Ion-Exchange ( c h a r g e ) - Hydrophobic I n t e r a c t i o n s - Reverse Phase - S i z e - E x c l u s i o n ( s i z e &. shape) - Chromatofocusing (pi) A f f i n i t y Interactions - Antibody - - Antigen - Hormone - - R e c e p t o r - Enzyme - - S u b s t r a t e / A n a l o g / I n h i b i t o r - Metal ion - - Ligand - Dye - - L i g a n d Characteristics - can be c o s t l y - powerful f o r Dilute/Low solutions

Purity

ISOLATION / RECOVERY. The g o a l here i s to c o n c e n t r a t e the s t a r t i n g s o l u t i o n ( e . g . , f e r m e n t a t i o n b r o t h ) , p r e f e r a b l y w i t h some degree o f purification. T h i s may or may not i n v o l v e c e l l breakage s i n c e the p r o d u c t s may be s e c r e t e d . H o m o g e n i z a t i o n has been found e f f e c t i v e f o r i n t r a c e l l u l a r p r o d u c t s , e s p e c i a l l y i n c l u s i o n b o d i e s , even though e x t r a c t i o n by b i o / c h e m i c a l means i s a f e a s i b l e o p t i o n . An advantage w i t h i n c l u s i o n b o d i e s i s t h a t they appear to be q u i t e s t u r d y and can withstand r a t h e r severe o p e r a t i n g c o n d i t i o n s . For s o l u b l e products (enzymes, p e p t i d e s , e t c . ) , however, c a u t i o n has to be taken to a v o i d d e n a t u r a t i o n and p r o t e o l y t i c c l i p p i n g s . Here speed and s o l u t i o n c o n d i t i o n s f o r minimizing p r o t e o l y t i c a c t i v i t y (temperature, pH, i o n i c s t r e n g t h , i n h i b i t o r s , e t c . ) are o f the e s s e n c e . Product s e c r e t i o n by g e n e t i c e n g i n e e r i n g means i s an a t t r a c t i v e approach due to the p o t e n t i a l p r o c e s s s i m p l i f i c a t i o n ( VO), p r o v i d e d t h a t comparable p r o d u c t i v i t y can be a c h i e v e d . Depending upon the n a t u r e o f the p r o d u c t a number o f l i q u i d / s o l i d s e p a r a t i o n methods can be used. For s e p a r a t i o n o f c e l l d e b r i s and p r o d u c t s i n s o l i d form ( e . g . , i n c l u s i o n b o d i e s , p r e c i p i t a t e d p r o d u c t s ) c e n t r i f u g a t i o n has emerged as the method o f c h o i c e i n which s i z e and d e n s i t y d i f f e r e n c e s between c e l l d e b r i s and p r o t e i n p a r t i c l e s are e x p l o i t e d (_11). The drawbacks here are h i g h c a p i t a l and o p e r a t i n g c o s t s . For some s i t u a t i o n s c r o s s - f l o w f i l t r a t i o n (UF/MF) may be a f e a s i b l e a l t e r n a t i v e . Aqueous two-phase e x t r a c t i o n , i n p a r t i c u l a r P E G / s a l t systems, has been s u c c e s s f u l l y a p p l i e d to c o n c e n t r a t i n g c e l l s and/or c e l l d e b r i s i n one phase and e x t r a c t i n g p r o d u c t s i n t o the o t h e r phase (12-13)· The advantage here i s t h a t not o n l y l i q u i d / s o l i d s e p a r a t i o n but a l s o c o n c e n t r a t i o n and p a r t i a l p u r i f i c a t i o n are a c c o m p l i s h e d at the same time. Compared w i t h o t h e r methods, a f a c t o r t h a t needs to be c o n s i d e r e d w i t h aqueous two-phase e x t r a c t i o n


2. HO Strategies for Large-Scale Protein Purification

23


i s the a d d i t i o n o f r e a g e n t s (polymers & s a l t s ) t o the system, which may c o m p l i c a t e downstream p r o c e s s i n g i f they have t o be removed a t the end. P a r t l y because o f t h i s v e r y i s s u e , l a r g e - s c a l e a p p l i c a t i o n s o f aqueous two-phase systems have been l i m i t e d t o p u r i f i c a t i o n o f i n d u s t r i a l enzymes where a c t i v i t y r a t h e r than a b s o l u t e p u r i t y i s needed . F o r s e c r e t e d p r o d u c t s o r i n t r a c e l l u l a r p r o d u c t s r e l e a s e d by p e r m e a b i l i z a t i o n , c e l l / l i q u i d s e p a r a t i o n f o l l o w e d by p r o d u c t c o n c e n t r a t i o n / d i a f i l t r a t i o n can be c o n v e n i e n t l y c a r r i e d out by cross-flow m i c r o f i l t r a t i o n or u l t r a f i l t r a t i o n . REFOLD. T h i s s t e p i s n e c e s s a r y i f the p r o t e i n i s i n an i n a c t i v e form ( d e n a t u r e d , r e d u c e d , e t c . ) . I t has been found t h a t o v e r e x p r e s s i o n o f f o r e i g n p r o t e i n s i n b a c t e r i a l systems o f t e n r e s u l t s i n formation o f i n c l u s i o n bodies. The p r o t e i n e x i s t s i n a r e d u c e d , p o l y m e r i c s t a t e which n e c e s s i t a t e s d i s s o l u t i o n and renaturation. T h i s s t e p i s q u i t e c r i t i c a l s i n c e i t not o n l y a f f e c t s the o v e r a l l y i e l d but through the n a t u r e o f the i m p u r i t i e s g e n e r a t e d w i l l a l s o d i c t a t e the subsequent p u r i f i c a t i o n t r a i n . For secreted p r o d u c t s , which tend t o be i n t h e i r a c t i v e form, r e f o l d i s not normally necessary. In the case o f i n c l u s i o n b o d i e s , t h e r e f o l d s t e p t y p i c a l l y c o n s i s t s o f f i r s t d i s s o l v i n g the s o l i d s i n a s t r o n g c h a o t r o p e such as g u a d i n i n e h y d r o c h l o r i d e , sodium t h i o c y a n a t e o r u r e a , f o l l o w e d by renaturing/oxidative r e f o l d i n g process. T h i s i s a complex p r o c e s s w i t h major y i e l d l o s s due t o a g g r e g a t i o n (_16). Key parameters f o r m i n i m i z i n g a g g r e g a t i o n o r m a x i m i z i n g the r e f o l d y i e l d a r e pH, temperature, time, and c o n c e n t r a t i o n o f dénaturants. A d d i t i v e s such as d e t e r g e n t s have a l s o been used ( 1 7 ) . The p r i n c i p l e a t work here seems t o be the d r i v e towards an o p t i m a l h y d r o p h o b i c / h y d r o p h i l i c b a l a n c e e x e r t e d on the p r o t e i n by i t s environment: s u f f i c i e n t h y d r o p h o b i c f o r c e i s needed t o cause the p r o t e i n t o r e f o l d but the same f o r c e a l s o l e a d s t o a g g r e g a t i o n . A great deal o f p r o p r i e t a r y information r e l a t e d to p r a c t i c a l r e f o l d i n g o f p r o t e i n s has been g e n e r a t e d i n the p r i v a t e s e c t o r s . C o n t r a r y t o t h e o r e t i c a l b e l i e f s , a number o f p r o t e i n s have been found t o r e f o l d q u i t e e f f i c i e n t l y (RF e f f i c i e n c y > 7 0 % ) a t h i g h c o n c e n t r a t i o n s ( g / L l e v e l ) u s i n g f a i r l y simple p r o c e d u r e s . It appears t h a t s i n c e p r o t e i n r e f o l d i n g i s not w e l l u n d e r s t o o d and s i n c e each p r o t e i n may be u n i q u e , g e n e r a l i z a t i o n t o the p o i n t o f e x c l u d i n g e x p e r i m e n t a l i n v e s t i g a t i o n may not be w a r r a n t e d a t t h i s time. GROSS PURIFICATION. The main purpose here i s t o remove as many i m p u r i t i e s as p o s s i b l e i n a simple s t e p o r s t e p ( s ) . F o r t h i s , b a t c h a d s o r p t i o n and p r e c i p i t a t i o n seem t o be most e f f e c t i v e . Batch adsorption i s p a r t i c u l a r l y e f f e c t i v e f o r dealing with d i l u t e s o l u t i o n s by s e l e c t i n g an a d s o r b e n t t h a t w i l l b i n d the p r o d u c t . S i n c e h i g h s e l e c t i v i t y i s not c r i t i c a l a t t h i s s t a g e , an a d s o r b e n t w i t h h i g h c a p a c i t y and some s p e c i f i c i t y f o r a p a r t i c u l a r p r o d u c t i s not t o o d i f f i c u l t t o f i n d . Common a d s o r b e n t s a v a i l a b l e c o m m e r c i a l l y are e i t h e r charged o r h y d r o p h o b i c . A f f i n i t y adsorbents using metal c h e l a t e o r dye l i g a n d s a r e v e r y e f f e c t i v e f o r group s p e c i f i c i n t e r a c t i o n s and s h o u l d be e x p l o i t e d when a p p r o p r i a t e . With


24



w e l l - t h o u g h t out a p p l i c a t i o n s , t h i s approach c o u l d a c c o m p l i s h c l a r i f i c a t i o n , c o n c e n t r a t i o n , and p a r t i a l p u r i f i c a t i o n i n p r a c t i c a l l y one i n t e g r a t e d s t e p , as i l l u s t r a t e d i n one o f the case studies discussed l a t e r . I f the s o l u t i o n i s f r e e o f p a r t i c u l a t e s , a d s o r p t i o n i n column mode can be done, which a l l o w s some l e v e r a g e f o r improved p u r i f i c a t i o n d u r i n g e l u t i o n . P r e c i p i t a t i o n can be a v e r y p o w e r f u l method f o r g r o s s purification. P r o t e i n p r e c i p i t a t i o n can be brought about by v a r i o u s means such as changes i n pH o r temperature o f the s o l u t i o n , by a d d i t i o n o f s a l t s ( e . g . ammonium s u l f a t e ) , w a t e r - s o l u b l e polymers (e.g. p o l y e t h y l e n e g l y c o l , p o l y e t h y l e n e imine, p o l y a c r y l i c a c i d ) , i n o r g a n i c f l o c c u l a n t s ( s i l i c a o r a l u m i n a , b i o p r o c e s s i n g a i d s from Rohm and Haas, c e l l d e b r i s remover from Whatman), o r o r g a n i c s o l v e n t s (e.g. a l c o h o l s ) . An e x c e l l e n t r e v i e w o f these p r e c i p i t a t i o n methods i s g i v e n by B e l l e t a l (_18). S a l t i n g out i n which molar c o n c e n t r a t i o n s o f s a l t s such as ammonium s u l f a t e are used i s a v e r y common method f o r p r o t e i n p r e c i p i t a t i o n . This approach, however, has many drawbacks: low s e l e c t i v i t y , h i g h s e n s i t i v i t y to o p e r a t i n g c o n d i t i o n s , and down stream c o m p l i c a t i o n s ( s a l t removal & d i s p o s a l ) . S i m p l e r and more e f f e c t i v e methods such as change i n pH o r temperature, use o f p o l y e l e c t r o l y t e s alone o r i n c o m b i n a t i o n w i t h n e u t r a l polymers ( 1 9 , 2 0 ) s h o u l d not be o v e r l o o k e d . There are two ways to use p r e c i p i t a t i o n . One convenient approach i s to p r e c i p i t a t e most o f the i m p u r i t i e s l e a v i n g the product i n s o l u t i o n f o r f u r t h e r p r o c e s s i n g . For t h i s , f l o c c u l a t i o n i s v e r y e f f e c t i v e . The p r e c i p i t a t e i s a network o f c e l l d e b r i s , e x t r a n e o u s p r o t e i n s , c o l o r e d c o n t a m i n a n t s , and, w i t h a n i o n exchange f l o c c u l a n t s such as p o l y e t h y l e n e i m i n e (2Λ) , n u c l e i c a c i d s . Instead o f p r e c i p i t a t i n g c o n t a m i n a n t s , i n some c a s e s i t may be e a s i e r to p r e c i p i t a t e the p r o d u c t . T h i s , however, would n e c e s s i t a t e s o l i d r e c o v e r y and r e d i s s o l u t i o n , which means a d d i t i o n a l p r o c e s s i n g s t e p s and y i e l d r e d u c t i o n . While the above methods appear common and tend not to be h i g h l y s e l e c t i v e , they can be v e r y e f f e c t i v e i f used a p p r o p r i a t e l y , e s p e c i a l l y f o r rDNA p r o d u c t s , because o f the f o l l o w i n g two r e a s o n s . F i r s t , most p r a c t i c a l rDNA p r o c e s s e s a c h i e v e f a i r l y h i g h e x p r e s s i o n ( 1 0 - 3 0 % o f t o t a l c e l l u l a r p r o t e i n s ) , so h i g h l y s o p h i s t i c a t e d t e c h n i q u e s are not u s u a l l y r e q u i r e d to i n c r e a s e the p u r i t y to 70-90%. Second, the p r o t e i n s o f i n t e r e s t b e i n g f o r e i g n to the b a c t e r i a l systems are l i k e l y to have v e r y d i f f e r e n t p r o p e r t i e s ( p i , h y d r o p h o b i c i t y , s i z e , heat s t a b i l i t y , e t c . ) t h a t s h o u l d be e x p l o i t e d f o r simple y e t e f f e c t i v e p u r i f i c a t i o n . T h i s w i l l be i l l u s t r a t e d i n one o f the case s t u d i e s . A n o t h e r e f f e c t i v e method f o r g r o s s p u r i f i c a t i o n i s l i q u i d - l i q u i d e x t r a c t i o n , e s p e c i a l l y aqueous two-phase systems (12-15)· These, however, have l i m i t e d a p p l i c a t i o n s so f a r because o f a number o f r e a s o n s : r e l a t i v e l y new method, h i g h polymer c o s t ( f o r PEG-dextran s y s t e m s ) , and the need to remove p h a s e - f o r m i n g r e a g e n t s ( p o l y m e r s , s a l t s ) from the p r o d u c t s . HIGH RESOLUTION PURIFICATION. V a r i o u s forms o f chromatography ( T a b l e IV) are p r e d o m i n a n t l y used f o r the f i n a l p u r i f i c a t i o n o f p r o t e i n s to homogeneity ( 2 2 ) . Ion-exchange chromatography i s w i d e l y used due to i t s v e r s a t i l e a p p l i c a b i l i t y f o r p r o t e i n s , h i g h c a p a c i t y



2.

HO

Strategiesfor Large-Scale Protein Purification

25

and r e s o l u t i o n . Based on l i t e r a t u r e i n f o r m a t i o n as w e l l as our e x p e r i e n c e , ion-exchange or r e v e r s e p h a s e / h y d r o p h o b i c i n t e r a c t i o n s chromatography can have remarkable r e s o l u t i o n d e s p i t e i t s s u p p o s e d l y n o n s p e c i f i c mode o f i n t e r a c t i o n s (charge o r h y d r o p h o b i c i t y ) . The n a t u r e o f the i n t e r a c t i o n between macromolecules ( w i t h t h e i r t h r e e - d i m e n s i o n a l s t r u c t u r e s ) and s u r f a c e s i s such t h a t unexpected s p e c i f i c i t y may r e s u l t from the dynamics o f m u l t i s i t e i n t e r a c t i o n s (23). With the l a r g e number o f parameters a v a i l a b l e f o r manipulation i n chromatographic s e p a r a t i o n — type o f r e s i n , degree o f l o a d i n g , washing c o n d i t i o n s , e l u t i o n s t r a t e g y ( s t e p , g r a d i e n t , d i s p l a c e m e n t o r c o m b i n a t i o n ; type o f b u f f e r o r s o l v e n t ) — r e s o l u t i o n o f m o l e c u l e s w i t h minor d i f f e r e n c e s i n amino a c i d r e s i d u e s o r between monomer and o l i g o m e r s c o u l d be a c h i e v a b l e . Very good r e v i e w a r t i c l e s and books can be found on the use o f chromatography f o r p u r i f i c a t i o n (24-26). Naveh, i n an e x c e l l e n t r e v i e w on s c a l e - u p s t r a t e g i e s f o r p r o t e i n p u r i f i c a t i o n ( 4 ) , o f f e r e d some p r a c t i c a l c o n s i d e r a t i o n s f o r u t i l i z i n g chromatography i n a p u r i f i c a t i o n scheme. He d i s c u s s e d the importance o f m a i n t a i n i n g c o n s i s t e n t f e e d s o l u t i o n ( e . g . p r o d u c t c o n c e n t r a t i o n and i m p u r i t y p r o f i l e ) due to o v e r l o a d i n g c o n d i t i o n s ; the s i g n i f i c a n c e o f dynamic c a p a c i t y as r e l a t e d to l i n e a r f l o w r a t e , s o l u t i o n pH and i o n i c s t r e n g t h ; and the r o l e o f m a t r i x p a c k i n g w i t h r e s p e c t to column p r e s s u r e drop through the c h r o m a t o g r a p h i c c y c l e . Chromatography u s i n g a f f i n i t y i n t e r a c t i o n s i s an important method but one has to be v e r y c a r e f u l i n the c h o i c e o f l i g a n d s . A n t i b o d i e s are g e n e r a l l y not a p r a c t i c a l c h o i c e due to h i g h c o s t , low c a p a c i t y , and f a i r l y poor s e l e c t i v i t y f o r c l o s e l y - r e l a t e d m o l e c u l e s (monomer/dimer, d e n a t u r e d , w r o n g l y - f o l d e d ) . Receptors appear to a b e t t e r c h o i c e w i t h r e g a r d s to these a s p e c t s , as found by Hoffmann La Roche i n the p u r i f i c a t i o n o f i n t e r f e r o n . M e t a l i o n s and dyes c o u l d be v e r y s e l e c t i v e f o r some p r o t e i n s , e s p e c i a l l y i f they are m o d i f i e d f o r enhanced i n t e r a c t i o n s . A unique a s p e c t o f a f f i n i t y methods i s t h a t , due to the s t r o n g i n t e r a c t i o n i n v o l v e d , they are a powerful t o o l f o r c o n c e n t r a t i o n / p u r i f i c a t i o n of d i l u t e s o l u t i o n s . As such, they c o u l d be viewed as c o n c e n t r a t i o n & g r o s s - p u r i f i c a t i o n methods, which need to be f o l l o w e d w i t h , f o r example, chromatography f o r the f i n a l p u r i f i c a t i o n . Even though t h i s o r d e r o f usage may seem s t r a n g e , i t makes sense i f one r e c o g n i z e s t h a t , due to l o c a l i z e d i n t e r a c t i o n s and the n a t u r e o f s i n g l e - s t a g e b i n d / r e l e a s e , most a f f i n i t y methods can not u s u a l l y s e p a r a t e s i m i l a r m o l e c u l e s . FINAL CONCENTRATION. At t h i s p o i n t i n the p r o c e s s , the u s u a l r e q u i r e m e n t i s p r o d u c t c o n c e n t r a t i o n w i t h o r w i t h o u t b u f f e r exchange ( e i t h e r f o r s t a b i l i t y and/or i n p r e p a r a t i o n f o r the f o r m u l a t i o n s t e p ) p r i o r to the d r y i n g s t e p . Both c o n c e n t r a t i o n and b u f f e r exchange can be c o n v e n i e n t l y c a r r i e d out i n one s t e p u s i n g ultrafiltration. F o r p r o d u c t s t h a t are not s t a b l e i n a c r o s s - f l o w f i l t r a t i o n environment ( h i g h c i r c u l a t i n g r a t e , l o n g p r o c e s s i n g time) o r t h a t have a s t r o n g tendency to f o u l membranes, the a l t e r n a t i v e i s g e l filtration. T h i s i s a m i l d e r method f o r b u f f e r exchange and removal o f s a l t s or low m o l e c u l a r weight c o n t a m i n a n t s ; i t i s , however, not s u i t a b l e f o r h a n d l i n g l a r g e volumes.


26



SOME PRACTICAL CONSIDERATIONS

RECOMMENDED INITIAL ACTIVITIES. Our e x p e r i e n c e shows t h a t the f i r s t c r i t i c a l s t e p i n the p r o c e s s development i s to e s t a b l i s h a r e l i a b l e assay f o r the p r o d u c t . Without t h i s , i t would be v e r y d i f f i c u l t t o a s s e s s the performance o f v a r i o u s methods and s e p a r a t i o n schemes. Next, one s h o u l d a l s o c h a r a c t e r i z e the s t a r t i n g s o l u t i o n i t s e l f — i n a d d i t i o n to the p u r i f i e d p r o d u c t , which i s commonly done. The purpose here i s t o look f o r main d i f f e r e n c e s between the p r o d u c t and c o n t a m i n a n t s , which w i l l serve as the b a s i s f o r d e v i s i n g a sound p u r i f i c a t i o n process. G e l e l e c t r o p h o r e s i s i s a q u i c k way to a s s e s s the s i z e (MW) and p u r i t y d i s t r i b u t i o n o f p r o t e i n s i n the m i x t u r e . P r o t e i n n e t charge and h y d r o p h o b i c i t y can be s t u d i e d w i t h a d s o r b e n t s ( a n i o n / c a t i o n , h y d r o p h o b i c ) o r aqueous two-phase systems. The i s o e l e c t r i c p o i n t s ( p i ) o f p r o t e i n s i n s o l u t i o n can be d e t e r m i n e d by i s o e l e c t r i c focusing. In a d d i t i o n , s o l u t i o n c h a r a c t e r i s t i c s such as s t a b i l i t y as a f u n c t i o n o f temperature and time, h a n d l i n g , e t c . , s h o u l d be c a r e f u l l y noted and taken i n t o account i n the p r o c e s s development. T h i s not o n l y w i l l minimize p r o c e s s i n g c o m p l i c a t i o n s a t the l a r g e - v o l u m e s t a g e but sometimes may a l s o o f f e r c l u e s f o r unique p u r i f i c a t i o n approaches.

SOME GUIDELINES FOR PURIFICATION PROCESS DEVELOPMENT Approaches f o r s e l e c t i o n o f e a r l y s t e p s i n the p u r i f i c a t i o n t r a i n : . reduce p r o c e s s volume e a r l y on . e l i m i n a t e components o f extreme n a t u r e : p a r t i c u l a t e s , small s o l u t e s , large aggregates, n u c l e i c a c i d s , e t c . A p p r o p r i a t e methods here a r e a d s o r p t i o n ( h y d r o p h o b i c o r i o n -exchange), p r e c i p i t a t i o n / f l o c c u l a t i o n , u l t r a f i l t r a t i o n , and a f f i n i t y adsorption. I n t e g r a t i o n o f P u r i f i c a t i o n Steps: . s t e p s s h o u l d be complementary t o one a n o t h e r both i n degree o f p u r i f i c a t i o n as w e l l as i n p r o c e s s flow t o a c h i e v e the f i n a l g o a l s . I t s h o u l d always be kept i n mind t h a t o p t i m i z a t i o n ( y i e l d , p u r i t y & c o s t ) i s done f o r the whole p r o c e s s not f o r any s i n g l e s t e p . T h i s i s to a v o i d b e i n g t r a p p e d i n t o u n d u l y m a x i m i z i n g a s t e p which may n o t make much d i f f e r e n c e i n the whole scheme. . minimize the number o f s o l v e n t s and b u f f e r s used. While t h i s may sound t r i v i a l and i s n o t t h a t c r i t i c a l a t the bench s c a l e , u n n e c e s s a r y s o l v e n t o r b u f f e r exchanges w i l l be c o s t l y and time consuming on a l a r g e - s c a l e . A l s o , avoid, i f p o s s i b l e , b u f f e r s that are expensive, c o m p l i c a t e d t o p r e p a r e , o r d i f f i c u l t to pH ( T r i s b u f f e r s ) Ease o f O p e r a t i o n : The key p o i n t here i s the s i m p l e r the p r o c e s s and the more s t r a i g h t f o r w a r d the c o n d i t i o n s , the l e s s l i k e l y f o r i t to f a i l on a l a r g e - s c a l e .


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27

CASE STUDIES


T a b l e V l i s t s the p o s s i b l e s o u r c e s o f p r o t e i n / p e p t i d e p r o d u c t s . Only a few r e p r e s e n t a t i v e r e a l examples a r e d i s c u s s e d here t o demonstrate the a p p l i c a b i l i t y o f the o u t l i n e d s t r a t e g i e s . MICROBIAL, INTRACELLULAR, INSOLUBLE (E.G., INCLUSION BODIES). An i n t e r e s t i n g case i n t h i s c a t e g o r y i s the p u r i f i c a t i o n p r o c e s s f o r f i b r o b l a s t b e t a - i n t e r f e r o n p r e s e n t e d by Hershenson o f Cetus Corp (9)· The i n t e r f e r o n was made i n the form o f i n c l u s i o n b o d i e s . The c e l l s were f i r s t homogenized to r e l e a s e the p r o d u c t , f o l l o w e d by c e n t r i f u g a t i o n t o b r i n g down the i n c l u s i o n b o d i e s l e a v i n g c e l l d e b r i s i n the s u p e r n a t a n t . The r e c o v e r e d i n c l u s i o n b o d i e s were then d i s s o l v e d and the p r o t e i n r e f o l d e d . I n the c l o n i n g o f i n t e r f e r o n , the odd c y s t e i n e r e s i d u e t h a t does not p a r t i c i p a t e i n the d i s u l f i d e f o r m a t i o n was d e l i b e r a t e l y r e p l a c e d w i t h s e r i n e t o m i n i m i z e m i s p a r i n g i n the r e f o l d i n g p r o c e s s . T h i s was done t o improve the refold efficiency. A f t e r the c o m p l e t i o n o f the r e f o l d s t e p , t h e r e are two t y p e s o f c o n t a m i n a n t s p r e s e n t : E. c o l i - d e r i v e d ( p r o t e i n s , e n d o t o x i n s , n u c l e i c a c i d s ) , and p r o d u c t - l i k e ( d i m e r / a g g r e g a t e s o f i n t e r f e r o n and i t s m o d i f i e d f o r m s ) . T h i s type o f m i x t u r e i s f a i r l y t y p i c a l f o r rDNA p r o d u c t s made by E. c o l i i n the form o f i n c l u s i o n bodies. An u n d i s c l o s e d " p r e t r e a t m e n t " s t e p ( g r o s s p u r i f i c a t i o n ?) was c a r r i e d o u t next to p r e p a r e a c l e a n e d - u p l o a d s o l u t i o n f o r the f i n a l p u r i f i c a t i o n s t e p i n which r e v e r s e phase chromatography was used t o s e p a r a t e i n t e r f e r o n from i t s o t h e r monomeric forms as w e l l as from i t s polymers. For the p u r i f i c a t i o n o f animal growth hormones, which f o l l o w s more o r l e s s the same scheme d i s c u s s e d above f o r b e t a - i n t e r f e r o n , we d i s c o v e r e d a v e r y p o w e r f u l p r e c i p i t a t i o n method f o r the g r o s s p u r i f i c a t i o n step ( 1 9 - 2 0 ) . B a s i c a l l y a n e u t r a l polymer and a charged polymer, both w a t e r - s o l u b l e , were used s i m u l t a n e o u s l y t o b r i n g about p r e c i p i t a t i o n o f almost a l l o f the c o n t a m i n a n t s ( F i g u r e s 4 & 5 ) . The r o l e o f the charged polymer i s t o form charged complex w i t h the c o n t a m i n a n t s . The n e u t r a l polymer enhances the p r e c i p i t a t i o n o f the complex as w e l l as improves the charged polymer specificity. Both p u r i t y and y i e l d i n e x c e s s o f 9 0 % c o u l d be achieved i n a s i n g l e p r e c i p i t a t i o n step. Chromatography c o u l d then be used f o r the f i n a l p u r i f i c a t i o n . MICROBIAL, EXTRACELLULAR, SOLUBLE. An example here i s the p u r i f i c a t i o n o f a p e p t i d e ( m o l e c u l a r weight about 3 0 0 ) made by a fungal fermentation. The b r o t h c o n t a i n e d a l o t o f f i n e p a r t i c l e s as w e l l as s t r i n g y , s l i m y s u b s t a n c e s . The p e p t i d e c o n c e n t r a t i o n and i t s p u r i t y i n the b r o t h were v e r y low ( F i g u r e 6 ) . Thus the s o l u t i o n needs t o be c l a r i f i e d and, as the p r o p o s e d s t r a t e g y i n d i c a t e s , s i g n i f i c a n t l y concentrated, p r e f e r a b l y with p a r t i a l p u r i f i c a t i o n , b e f o r e a h i g h - r e s o l u t i o n p u r i f i c a t i o n method i s used. By s c r e e n i n g the recommended i s o l a t i o n and g r o s s p u r i f i c a t i o n methods ( T a b l e I &, I I I ) we were a b l e to q u i c k l y d e v e l o p a v e r y e f f i c i e n t , e a s i l y s c a l a b l e p r o c e s s i n which a simple a d s o r p t i o n / e x t r a c t i o n s t e p was used t o a c h i e v e s u b s t a n t i a l c o n c e n t r a t i o n and g r o s s p u r i f i c a t i o n . Shown i n F i g u r e 7 , the b r o t h was f i r s t s u b j e c t e d to a c o a r s e f i l t r a t i o n s t e p m a i n l y t o remove l a r g e p a r t i c l e s and s t r i n g y


28


TABLE V. 1)

MICROBIAL, INTRACELLULAR, INSOLUBLE e.g.


SOURCES OF PROTEINS AND PEPTIDES

i n s u l i n , a n i m a l growth hormones, b e t a - i n t e r f e r o n AP3-Rec A f u s i o n p r o t e i n , i n t e r l e u k i n - 2

C h a r a c t e r i s t i c s : i n c l u s i o n or r e f r a c t i l e bodies p r o d u c t i n r e d u c e d , a g g r e g a t e d forms high product c o n c e n t r a t i o n 2)

MICROBIAL, INTRACELLULAR, SOLUBLE e.g.

L-aspartase, factor

human growth hormone, tumor

necrosis

C h a r a c t e r i s t i c s : products s u s c e p t i b l e to p r o t e o l y t i c d e g r a d a t i o n and h i g h l y c o n t a m i n a t e d w i t h s o l u b l e c e l l u l a r components upon cell lysis. 3)

MICROBIAL, EXTRACELLULAR (SOLUBLE) e.g.

4)

IGF-1,

TISSUE CULTURE e.g.

detergent

enzymes, r e n n i n

(EXTRACELLULAR & SOLUBLE)

t-PA, monoclonal a n t i b o d i e s ,

interleukin-4

C h a r a c t e r i s t i c s : v e r y low p r o d u c t c o n c e n t r a t i o n major c o n t a m i n a n t s : serum p r o t e i n s o r p r o t e i n a d d i t i v e s (BSA, e t c . ) 5)

PEPTIDE SYNTHESIS e.g.

AP3

C h a r a c t e r i s t i c s : no m i c r o b i a l p r o t e i n s / e n d o t o x i n s high product concentration c l o s e a n a l o g s as major i m p u r i t i e s


HO



100

DEAE-DEXTRAN LEVEL (g / g proteins) F i g u r e 4. The use o f PEG and DEAE-dextran f o r p r o t e i n p r e c i p i t a t i o n from a f e r m e n t a t i o n b r o t h c o n t a i n i n g an animal growth hormone.




Figure 5 . SDS-PAGE o f E. c o l i crude e x t r a c t (Lane 1 ) and a f t e r p r e c i p i t a t i o n w i t h v a r i o u s c o m b i n a t i o n s o f n e u t r a l and charged polymers (Lane 2 - 8 ) . Dark bands at bottom are b o v i n e growth hormone.




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31

32


Fermentation Broth ι Coarse Filtration


Batch Adsorption ι Filtration / Wash

Extraction

clear solution 10x

concentration

70-80% yield significant purification

Chromatography 99%

purity

τ Figure

7.

P u r i f i c a t i o n process developed

f o r the

fungal

peptide.

materials. A h y d r o p h o b i c r e s i n (XAD s e r i e s from Rohm & Hass) was then added d i r e c t l y to the c l o u d y f i l t r a t e to a d s o r b the p e p t i d e as w e l l as some o t h e r components. The subsequent s i m p l e f i l t r a t i o n / w a s h s t e p removed a l l the f i n e p a r t i c u l a t e s i n s o l u t i o n by e x p l o i t i n g the l a r g e s i z e d i f f e r e n c e between the r e s i n and the particulates. E x t r a c t i o n o f the r e s i n r e s u l t e d i n a c l e a r s o l u t i o n t h a t was 1 0 - f o l d c o n c e n t r a t e d w i t h 7 0 - 8 0 % y i e l d and s u b s t a n t i a l peptide p u r i f i c a t i o n (Figure 8 ) . The f i n a l p u r i f i c a t i o n u s i n g chromatography to get to 9 9 % p u r i t y was g r e a t l y s i m p l i f i e d owing to the r e l a t i v e l y pure and c o n c e n t r a t e d l o a d s o l u t i o n . TISSUE CULTURE (EXTRACELLULAR, SOLUBLE). For p u r i f y i n g p r o t e i n s made by t i s s u e c u l t u r e ( e . g . , m o n o c l o n a l a n t i b o d i e s , t-PA) S c o t t and coworkers at I n v i t r o n ( 2 7 ) s u g g e s t e d s e v e r a l schemes i l l u s t r a t e d i n F i g u r e 9 f o r c o n d i t i o n e d media. T h e i r recommended scheme ( # 2 ) i s more o r l e s s c o n s i s t e n t w i t h our p r o p o s e d s t r a t e g y . A key c h a r a c t e r i s t i c o f t i s s u e c u l t u r e i s t h a t p r o d u c t l e v e l s tend to be q u i t e low. C o n c e n t r a t i o n i s thus a n e c e s s a r y f i r s t s t e p , f o r which u l t r a f i l t r a t i o n comes i n handy. For v e r y d i l u t e s o l u t i o n s , however, a f f i n i t y a d s o r p t i o n may have to be used a l s o f o r the concentration s t e p to m i n i m i z e p r o d u c t l o s s due to n o n s p e c i f i c b i n d i n g and l o n g p r o c e s s i n g time a s s o c i a t e d w i t h u l t r a f i l t r a t i o n . For media c o n t a i n i n g serum p r o t e i n s , p r e c i p i t a t i o n c o u l d be an e f f e c t i v e and simple g r o s s p u r i f i c a t i o n method f o r removing t h e s e p r o t e i n s b e f o r e moving on to chromatography.



HO

33

Strategiesfor Large-Scale Protein Purification

ο CO CO

Protein Purification - ACS Publications - American Chemical Society

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