Recovery of Proteins from Polyethylene Glycol ... - ACS Publications

8 Recovery of Proteins from Polyethylene Glycol-Water Solution by Salt Partition G. B. Dove and G. Mitra

Downloaded by MONASH UNIV on April 14, 2016 | http://pubs.acs.org Publication Date: July 11, 1986 | doi: 10.1021/bk-1986-0314.ch008

Cutter Laboratories, Berkeley, CA 94710

Addition of salts (e.g. potassium phosphate dibasic) partitions an aqueous system containing 20% w/v polyethylene glycol (PEG) into two liquid phases: a PEG enriched phase and a salt enriched phase. Proteins and polymers (e.g. DNA, albumin, immunoglobulins, alpha-1 antitrypsin, and PEG) distribute unevenly between the two phases. Partition coefficients (concentration in PEG phase / concentration in salt phase = K) are influenced by physical parameters, such as salt composition and concentration, pH (ion ratios) and temperature. Specific proteins (e.g. alpha-1 antitrypsin) exhibit low Κ values in a wide range of conditions. Higher salt concentrations and pH yield higher partition coefficients. In a plasma source, the Κ of alpha-1 antitrypsin is 0.0006 at 0.5 M salt and increases to 0.0062 at 1.6 M salt. The Κ of PEG increases to 200+ in 1.0 M salt. Proteins in general exhibit Κ values of 0.01-100. Altering pH to make proteins or other partitioned materials more/less hydrophilic induces greater/lower solubility. A pH change from 5 to 9 increases Κ in general by 100-fold+. Further, the trends demonstrated by increasing salt concentration are amplified. Lower temperatures (5 to -5 C) increase the PEG Κ by two to ten-fold with little change in protein distribution. Conditions may be tailored to optimize isolation of specific proteins to permit recoveries of 90% from mixed systems, such as plasma or fermentation broths. T h i s paper i s o r g a n i z e d i n t o t h r e e p a r t s . P u r i f i c a t i o n techniques a r e o u t l i n e d b r i e f l y i n c o m p a r i s o n t o aqueous e x t r a c t i o n , followed by a review o f p r o p e r t i e s and work i n m u l t i p h a s e systems with emphasis on t h e p u r i f i c a t i o n o f p r o t e i n s . Finally, recent work 0097-6156/ 86/ 0314-0093S06.00/ 0 © 1986 American Chemical Society

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


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SEPARATION, RECOVERY, AND PURIFICATION IN BIOTECHNOLOGY

undertaken by the authors i s presented, involving proteins of pharmaceutical importance. Various methods are available f o r the separation of biochemicals. These i n c l u d e p h y s i c a l methods o f c e n t r i f u g a t i o n and filtration, c h e m i c a l methods o f p r e c i p i t a t i o n and e x t r a c t i o n , and i n t e r a c t i v e t e c h n i q u e s , s u c h a s e l e c t r o p h o r e s i s and chromatography. T h e s e methods a r e employed t o p e r f o r m t h e s t e p s n e c e s s a r y t o p u r i f y biological m a t e r i a l s from complex s o l u t i o n s . The i s o l a t i o n o f a specific component ( e . g . a p r o t e i n ) from a plasma s o u r c e or a fermentation broth r e q u i r e s s e v e r a l stepss a) removal of c e l l particles (disruption i f intracellular p r o d u c t ) , e.g. c e n t r i f u g a t i o n . b) p r e l i m i n a r y p u r i f i c a t i o n , e.g c o n c e n t r a t i o n , p r e c i p i t a t i o n . c) s e c o n d a r y p u r i f i c a t i o n , e.g. high-résolut!on chromatography. d) finishing. Through these steps, the necessary p u r i t y and yield are achieved. Requirements f o r p u r i t y and y i e l d a r e d i c t a t e d by f i n a l use. P u r i t y may range as low a s 10% f o r b u l k enzymes t o v i r t u a l l y 100% f o r t h e r a p e u t i c use. The f i n a l y i e l d a f f e c t s directly the cost of the finished product and i s of c r i t i c a l economic importance, as feed stocks f o r the processes are typically expensive. Final y i e l d s a r e c o n s i d e r e d i n terms o f biologically active material, as many o f t h e components a r e l a b i l e and u s e l e s s i n a denatured s t a t e . S p e c i f i c a l l y , the c o n s t r a i n t s of high p u r i t y and biologically-active y i e l d i n the production of therapeutic products limit the alternatives available for purification processes. Extractions and precipitations i n chemistry are welle s t a b l i s h e d f o r o r g a n i c systems. F o r example, n u c l e i c a c i d s may be extracted in a phenol/water m i x t u r e (J.). The use of aqueous e x t r a c t i o n s has s e v e r a l advantages o v e r t h e s e and o t h e r w i d e l y used methods o f s e p a r a t i o n . 1) C h e m i c a l components, s u c h as polymers and/or salts, may be chosen t o m i n i m i z e dénaturât!on due to solvency or i n t e r f a c i a l tension ( 2 ) . Solvent/water m i x t u r e s , such as phenol/water, produce i n t e r f a c i a l t e n s i o n s i n t h e range o f 50 dyne/cm, compared to 0.1 dyne/cm i n aqueous s y s t e m s ^ ) . 2) Physical sources o f dénaturât!on a r e m i n i m a l , with v i r t u a l l y no shear. S i m p l e m i x i n g o n l y i s r e q u i r e d ; c e n t r i f u g a t i o n may be used to hasten separation (4). 3) C o n d i t i o n s may be tailored to satisfy s p e c i f i c i s o l a t i o n r e q u i r e m e n t s by b a s i n g s e p a r a t i o n s on dissimilar s o l u b i l i t i e s and a f f i n i t i e s , which a r e dependent on pH and s a l t s . These e f f e c t s a r e not a p p l i c a b l e t o o t h e r methods. 4) The process i s e a s i l y s c a l e d t o any volume o f m a t e r i a l , with minimal c a p i t a l i n v e s t m e n t 4 ) . Beyond c o n v e n t i o n a l p r o d u c t s , the technique i s applicable to biotechnical separations with unique p o s s i b i l i t i e s , which w i l l be a d d r e s s e d below. P r o p e r t i e s and A p p l i c a t i o n s o f Aqueous Systems The methodology of aqueous e x t r a c t i o n s i s a d a p t a b l e to the requirements o f i s o l a t i n g b i o l o g i e s due t o t h e h i g h water content of the system. The a d d i t i o n o f w a t e r - s o l u b l e polymers and/or salts t o water produces s p o n t a n e o u s l y two o r more l i q u i d phases. The d e n s e r s o l u t i o n ( u s u a l l y t h e s a l t - r i c h one) forms t h e bottom phase. Each phase i s c o m p r i s e d p r i m a r i l y o f w a t e r (80-95%X.2).



8.

DOVE A N D M I T R A

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Recovery of Proteins by Salt Partition

The t o p and bottom l i q u i d r e g i o n s a r e s e p a r a t e d by an i n t e r f a c e (P1). P r e c i p i t a t e s may form a t t h i s l i q u i d - l i q u i d i n t e r f a c e o r may settle t o t h e bottom o f t h e v e s s e l ). A p r o t e i n i n t h e system will d i s t r i b u t e between t h e two phases a c c o r d i n g t o t h e p r o p e r t i e s of the partitioning a g e n t s and o t h e r m a t e r i a l s present. A p a r t i t i o n c o e f f i c i e n t ( K ) may be d e f i n e d a s ( 2 ) s Κ

=

Ct/Cb

Salt ConcQntration (M) Figure 2. Albumin p a r t i t i o n c o e f f i c i e n t (concentration i n PEG phase / c o n c e n t r a t i o n i n s a l t phase) a s a f u n c t i o n o f s a l t concentration.


SEPARATION, RECOVERY, A N D PURIFICATION IN BIOTECHNOLOGY


100

Figure 4. Immunoglobulin G function of salt concentration.

partition

coefficient


as

a


8.


-

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Recovery of Proteins by Sait Partition

DNA

Figure 6. Summary o f p a r t i t i o n c o e f f i c i e n t s function of s a l t concentration.

a t pH 8 - 9 a s


a

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•

AlbiMin

Salt Concentration (M) Figure 7. Summary o f p a r t i t i o n c o e f f i c i e n t s function of s a l t concentration.

a t pH 5-6 as


a

8.


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Recovery of Proteins by Salt Partition

Table I I . Concentration of materials i n the s a l t phase and p a r t i t i o n c e f f i c i e n t a s f u n c t i o n s o f s a l t c o n c e n t r a t i o n and pH. Material t= 5 C

Salt

Cone*η (M)

PH

0.5 0.8 0.8 0.8 1.6

9 6 7 9 9

0.5 0.8 0.8 0.8 1.6

9 6 7 9 9

0.5 0.6 0.8 1.0 1.2 1.6

8 8 8 8 8 8

DNA


IgM

Alpha-1

P a r t i t i o n Coeff. Cone *n ( S a l t phase) ( P E G / S a l t ) (ug/ml ) 23.5 337 5.6 5.4 1 ( mg/ml ) 1.0 1.4 0.080 0.035 0.0019 ( mg/ml ) 17.8 10.1 6.3 5.5 4.7 3.3

0.043 0.004 0.179 0.185 538 0.0019 0.0013 0.024 0.054 n.a. 0.00056 0.0025 0.0033 0.0038 0.0044 0.0062

The d i f f e r e n c e between alpha-1 and o t h e r p r o t e i n s may be attributed t o e i t h e r t h e i n t r i n s i c n a t u r e o f alpha-1 (e.g. low hydrophobicity) o r t h e p r e s e n c e o f m i s c e l l a n e o u s plasma fraction contaminants i n t h e s a l t phase a t t r a c t i n g alpha-1 o r i n t h e PEG phase r e p e l l i n g alpha-1. A mixture of albumin and IgM show q u a l i t a t i v e l y t h e same v a l u e s a s t h e r e s p e c t i v e s i m p l e systems. As the s a l t concentration increases, the concentration of protein i n the s a l t phase d e c r e a s e s and t h e p a r t i t i o n c o e f f i c i e n t increases. Again, a t l o w e r pH, m a t e r i a l s do n o t m i g r a t e t o t h e PEG phase and partition coefficients do n o t exceed 1. Further work i s i n p r o g r e s s t o d e f i n e t h e s e p a r a t i o n between m u l t i p l e components based on e x p e r i m e n t s w i t h d e f i n e d systems. The effects o f s a l t c o n c e n t r a t i o n and pH have been studied p r e v i o u s l y (2j. 16, 3 2 ) . I t h a s been found t h a t t h e c o n c e n t r a t i o n and pH a r e n o t a s c r i t i c a l a s t h e r a t i o o f i o n s . The pH i s a measure o f t h e i o n i c environment; t h a t i s , t h e r a t i o o f charged i o n s d e r i v e d from t h e s a l t , K2HP04 and a c i d , H3P04. Compared t o effects o f small ions, t h e c o n c e n t r a t i o n o f p r o t e i n s has l i t t l e effect on p a r t i t i o n c o e f f i c i e n t s ( 3 2 ) . I n general, higher valent anions yield higher partition coefficients: Κ o f (P04)3- > (HP04)2- > (H2P04)-. The e f f e c t s of cations and a n i o n s a r e cumulative. Ionic effects c a n i n c l u d e t h e a d d i t i o n o f NaCl t o PEG/dextran systems ( 2 ) . The d i s t r i b u t i o n may be d e f i n e d i n terms o f a model From t h e B r o n s t e d f o r m u l a

Recovery of Proteins from Polyethylene Glycol ... - ACS Publications

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