A System for Heparin Removal - Advances in Chemistry (ACS

31 A System for Heparin Removal R. LANGER Massachusetts Institute of Technology, Department of Nutrition and Food Science, Cambridge, MA 02139 and Children's Hospital Medical Center, Department of Surgery, Boston, MA 02115 R. J. LINHARDT, P. M . GALLIHER, M. M. FLANAGAN, and C. L. COONEY Massachusetts Institute of Technology, Department of Nutrition and Food Science, Cambridge, MA 02139 M. D. KLEIN University of Michigan, Section of Pediatric Surgery, Ann Arbor, MI 48109

Extracorporeal rinization

medical machines rely on systemic hepa-

to improve blood compatibility. However, heparin

can lead to serious complications such as hemorrhaging. We propose a new approach to control heparin levels by employing a blood filter containing immobilized heparinase. Such a filter could potentially enable heparinization

of an extracorporeal

circuit without simultaneous heparinization of the patient. The principal findings of our work thus far include (1) increasing volumetric enzyme production over 1000-fold from previously published

procedures;

(2)

purifying

heparinase

by over

1000-fold from the crude cell extracts; (3) characterizing

the

biochemical properties of heparinase; (4) isolating the first heparinase inhibitors; (5) immobilizing heparinase with 91% activity recovery and excellent stability; and (6) demonstrating that columns as small as 1.5 mL can remove clinically used quantities of heparin in aqueous medium and in blood.

E

xtracorporeal medical machines (e.g., artificial kidney, pump-oxygenator) perfused with blood have been an effective part of the therapeutic arma-

mentarium for many years. These devices all rely on systemic heparinization to provide blood compatibility. Despite continuous efforts to improve anti-

coagulation techniques, many patients still develop coagulation abnormalities with the use of these devices (1-3). Even longer perfusion times may occur with machines such as the membrane oxygenator. In such cases, the drawbacks of systemic heparinization are multiplied (4). A number of ap0065-2393/82/0199-0493$06.00/0 ©1982 American Chemical Society

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BIOMATERIALS: INTERFACIAL PHENOMENA AND APPLICATIONS

proaches have b e e n a t t e m p t e d to solve this p r o b l e m . T h e s e i n c l u d e : (1) a d m i n i s t r a t i o n o f c o m p o u n d s to n e u t r a l i z e h e p a r i n (5); (2) d e v e l o p m e n t of heparin substitutes (6); (3) b o n d i n g of h e p a r i n (7-12) or other substances (13) to the extracorporeal d e v i c e ; a n d (4) d e v e l o p m e n t of n e w b l o o d - c o m p a t i b l e materials for c o n s t r u c t i o n o f the extracorporeal device (14). I n spite of these efforts, h e p a r i n i z a t i o n continues to be u s e d extensively i n a l l extracorporeal treatments, a n d c o n t r o l of b l o o d h e p a r i n levels remains a serious p r o b l e m . W e propose a n e w a p p r o a c h that w o u l d allow the f u l l h e p a r i n i z a t i o n of the extracorporeal d e v i c e , yet c o u l d enable, o n - d e m a n d , e l i m i n a t i o n of hepa r i n i n the patient's b l o o d s t r e a m . T h i s approach consists of a b l o o d filter c o n t a i n i n g i m m o b i l i z e d heparinase, w h i c h c o u l d be p l a c e d at the effluent o f any extracorporeal d e v i c e ( F i g u r e 1). S u c h a filter c o u l d theoretically be used to e l i m i n a t e h e p a r i n after it h a d served its p u r p o s e i n the extracorporeal device a n d before it r e t u r n e d to the patient. I n this chapter we discuss o u r efforts to d e v e l o p such a filter. O u r w o r k has focused o n several areas: (1) e n z y m e p r o d u c t i o n ; (2) e n z y m e p u r i f i c a t i o n ; (3) characterization of hepa rinase; (4) i m m o b i l i z a t i o n of heparinase; a n d (5) i n v i t r o testing of i m m o b i l i z e d heparinase.

Experimental Materials. Heparin, as the sodium salt, from porcine intestinal mucosa, was purchased from Sigma Chemical Co. (Grade II, 153 USP k units). Azure A dye was purchased from Fisher Scientific Co. (A-970, certified biological stain, total dye content 70%). The following polymer supports used in the immobilization were obtained preactivated: (1) Sepharose-CNBr 4B from Pharmacia; (2) polyacrylamide polyacetyl and enzacryl A H from Aldrich Chemical Co.; and (3) polyacrylamide NHS active ester (PAN 1000) as a gift from George M . Whitesides, MIT, Department of Chemistry. The unactivated polymer supports were obtained from the following sources: (1) poly(2-hydroxyethyl methacrylate) (PHEMA) from Polysciences Inc. and P H E M A (Spheron) from Hydron Inc. ; (2) Dacron (poly(ethyleneterephthalate)) from Aldrich Chemical Company; (3) poly(methyl (methacrylate) (PMA) 7% divinyl benzene crosslinked) from Rohm and Haas Company; and (4) silicone (Masterflex silicone tubing) from Cole-Parmer. The activating agents used were: (1) cyanogen bromide (CNBr), hexamethylene diisocyanate, and Woodwards Κ reagent from Aldrich Chemical Com pany; (2) l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) from CalbiochemBehring Corporation; (3) glutaraldehyde (EM grade 25%) from Polysciences, Inc.; and (4) organosilane ester (A-1100) from Union Carbide Corporation, Silicones division. The heparinase inhibitors were purchased from the following sources: (1) poly v i n y l sulfate) from Sigma Chemical Company; (2) polyanethole sulfonate from Calbiochem Corporation; and (3) polystyrene sulfonate (aqueous solution MW 70,000) from Polysciences Inc. The purification used hydroxylapatite (HTP) from Biorad Inc., protamine sulfate, bovine serum albumin (BSA), and polylysine (Type VI) from Sigma Chemical Com pany, and epoxy-activated Sepharose from Pharmacia Inc. Sodium dodecyl sulfate (SDS) gel electrophoresis and isoelectric focusing (IEF) were performed using chem icals and equipment from Biorad Inc.

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System for Heparin Removal

495

PATIENT

Figure 1. Proposed heparin circuit. The extracorporeal device could be a renal dialysis unit or a pump-oxygenator. The heparinase reactor could be part of a blood filter to be used either continuously (in which case heparin would be added continuously at the start of the circuit) or at the end of an operation. Heparin could thus be confined to the extracorporeal circuit.

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BIOMATERIALS: INTERFACIAL P H E N O M E N A A N D APPLICATIONS

Chondroitinase ABC from Proteus vulgaris was purchased from Sigma Chemical Company. All inorganic chemicals were reagent grade. All chemicals used in heparinase production are as described previously (15). Analytical Determinations. P R O T E I N C O N T E N T . Protein was measured by the method of Lovvry (16). H E P A R I N A S E ACTIVITY. Several assays were used to follow heparinase activity. These assays followed (1) the disappearance of heparin, (2) the appearance of heparin degradation products, or (3) the loss of the physiological function of heparin in anticoagulation. The basis of these assays and explanations as to when they are routinely used are listed below. 1. Metachromasia in Azure A. This assay measures heparinase activity by following the disappearance of heparin. Jacques (17) proposed that Azure A dye molecules dimerize in the presence of heparin, resulting in a decrease in the ττ-delocalization. This effect is observed as a shift in the absorption maximum from λ max = 620 nm to λ max = 520 nm. Since heparinase cleaves the α-linkage of heparin, its action causes chain shortening, resulting in less metachromasia. The presence of heparin or heparin-derived polysaccharide chains of hexasaccharide or larger (18) can be measured easily and reproducibly at levels of 1-10 μg/mL in crude (i.e., fermentation broths and cell sonicates) and purified preparations. Experimental details for utilizing this assay were reported in a previous publication (15). 2. a. Reducing Sugar Assay. With each cleavage of the heparin chain by hepa rinase (which is an α-eliminase), one reducing end group is formed and one aβ-unsaturated end group. The increase in reducing capacity, therefore, gives a mea sure of enzyme activity and product formation. This assay has been used routinely in assaying crude preparations of heparinase. The reducing capacity of the products produced from heparin at different stages in the purification of heparinase is variable. For example, the reducing capacity of products produced by the action of purified heparinase is lower than that obtained by the action of crude heparinase preparations. This result is apparently due to enzymes in crude preparations that cause further degradation of the products formed by heparinase. The Park and Johnson method (19) of measuring reducing sugars was used throughout these studies. b. Ultraviolet Assay. The α-β-unsaturated acid end group resulting from heparin cleavage is a chromophore with a λ max at approximately 232 nm and a molar absorptivity of about 5 x 10° M (20). The action of heparinase can be measured by sampling the reaction mixture, quenching it in 0.03N hydrochloric acid, and mea suring the absorbance. This assay can only be used to measure activity in relatively pure preparations due to two factors: (1) high concentration of protein interferes with the measurement of product, and (2) contaminating enzymes are capable of acting on the heparinase-derived product, resulting in the loss of the chromophore being measured. Because of its accuracy and ease of performing, this assay was the method of choice for measuring heparinase activity in the more purified heparinase preparations. 3. Assays Measuring the Loss of Heparins Biological Activity. The loss of heparin's biological activity through the action of heparinase can be tested by a number of available assays. Three assays were chosen for their ease of use and the range of activity they measure. Whole blood recalcification time (21) involves the measurement of heparin in citrated whole blood by recalcifying the blood and mea suring the clotting time. Factor X Assay (22) involves measuring the heparin in citrated plasma which has been enriched in Factor X by recalcifying and measuring the clotting time. Thrombin-antithrombin time (23) determines the action of heparin on the thrombin-antithrombin interaction by measuring the appearance of the chro mophore released by thrombin from a synthetic polypeptide substrate. In each of these assays a standard curve was constructed with each determination. 1

a

a

31.

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497


Results Heparinase Production.

T h e objective o f these studies was to develop

an u n d e r s t a n d i n g o f w h a t factors i n f l u e n c e heparinase p r o d u c t i o n b y Flavobacterium heparinum (15). T h i s was done b y s t u d y i n g the kinetics o f m i c r o bial g r o w t h a n d heparinase p r o d u c t i o n , a n d b y d e v e l o p i n g a s i m p l e , d e f i n e d m e d i u m to support this g r o w t h a n d p r o d u c t i o n . A 1000-fold increase i n v o l u m e t r i c heparinase p r o d u c t i o n over p r e v i o u s l y r e p o r t e d results was o b tained b y i m p l e m e n t i n g i m p r o v e d techniques o f m i c r o b i a l cultivation a n d environmental a n d genetic manipulations. These improvements and our findings are s u m m a r i z e d b e l o w . T h e w i l d t y p e strain o f Flavobacterium heparinum produces a n o n extracellular heparinase i n the g r o w t h stage o n l y w h e n h e p a r i n is s u p p l i e d to the g r o w t h m e d i u m as an i n d u c e r (25). E n z y m e p r o d u c t i o n occurs d u r i n g growth, so factors affecting g r o w t h can d i r e c t l y affect e n z y m e p r o d u c t i o n . A reliable heparinase p r o d u c t i o n s c h e m e was first w o r k e d out b y g r o w i n g the bacteria i n a c o m p l e x p r o t e i n digest m e d i u m . I n d u c e r was p r o v i d e d at the t i m e o f i n o c u l a t i o n o f the sterile m e d i u m . G r o w t h was i n i t i a l l y exponential, and h e p a r i n was r a p i d l y taken u p b y the c e l l at a rate of 1.1 g/g c e l l - h ( F i g u r e 2). E n z y m e specific activity began to increase just as h e p a r i n uptake was f i n i s h i n g , a n d increased at a v o l u m e t r i c rate o f 375 units/L-h. A t the onset of the stationary c e l l g r o w t h phase, e n z y m e p r o d u c t i o n stopped, and a deactivation was o b s e r v e d , r e s u l t i n g i n an 8 6 % loss o f total activity w i t h i n 4 h . To avoid this deactivation, the kinetics o f e n z y m e p r o d u c t i o n had to be u n d e r stood. T i m e l y harvest was thus i m p o r t a n t to obtain h i g h l y active heparinase. F i f t e e n fermentations w e r e p e r f o r m e d , a l l y i e l d i n g a total e n z y m e level o n the average of 9600 units o f heparinase/L o f f e r m e n t o r b r o t h , demonstrating the r e l i a b i l i t y o f this m e t h o d . To u n d e r s t a n d b e t t e r the e n v i r o n m e n t a l factors g o v e r n i n g e n z y m e p r o d u c t i o n , a d e f i n e d g r o w t h m e d i u m was d e v e l o p e d . T h i s m e d i u m was the result of n u t r i t i o n a l r e q u i r e m e n t e x p e r i m e n t s p e r f o r m e d to elucidate the growth factors r e q u i r e d b y this b a c t e r i u m . T h e b a c t e r i u m was a h i s t i d i n e auxotroph w i t h an a d d i t i o n a l (though not obligate) r e q u i r e m e n t for m e t h i onine. N o v i t a m i n r e q u i r e m e n t was observed. T h i s result p e r m i t t e d the use of the f o l l o w i n g d e f i n e d g r o w t h m e d i u m : glucose (main carbon source), heparin

(inducer),

(NH ) S0 4

2

4

(nitrogen

source),

K HP0 , 2

4

Na HP0 , 2

4

L - h i s t i d i n e , L - m e t h i o n i n e , trace salts, and M g S 0 - 7 H 0 . A 3 0 % increase i n 4

2

growth rate was o b s e r v e d u s i n g this m e d i u m . V o l u m e t r i c heparinase p r o d u c t i o n was increased fourfold over the c o m p l e x m e d i u m p r o d u c t i o n to 1480 units/L-h. A d d i t i o n a l l y , h i g h e r c e l l densities w e r e obtained r o u t i n e l y i n this d e f i n e d m e d i u m . A typical p r o d u c t i o n r u n u s i n g 20 g/L of glucose results i n a tenfold increase i n total e n z y m e o b t a i n e d to 96,000 units/L f e r m e n t o r b r o t h . T h i s f e r m e n t a t i o n has b e e n repeated eight times to date, d e m o n s t r a t i n g the r e l i a b i l i t y o f this m e t h o d . I n a d d i t i o n , the p r o d u c t heparinase is

498


>

ο < ο LL Ο ω ο. to Lu

Φ Ο λ

α.

σ> Ε \ Φ

•σ σ ». σ» Φ

< ζ φ or Χ

έ

Lu ε I

Figure 2. Results of a typical fermentation on complex medium showing heparin (O), heparinase specific activity (\Z\)> d dry cell weight (A) as a function of time as determined in a 2-L fermentor (15). an

more stable, since no r a p i d loss of the e n z y m e occurs i n this m e d i u m ( F i g u r e 3), and allows m o r e f l e x i b i l i t y a n d r e l i a b i l i t y for p r o d u c t recovery. T h e use of this d e f i n e d m e d i u m also p e r m i t t e d tests c o n c e r n i n g the effect o f m e d i u m components a n d e n v i r o n m e n t a l factors o n e n z y m e p r o d u c t i o n . O p t i m u m initial glucose a n d ( N H ) S 0 concentrations w e r e 8 g/L and 0.5 g/L, re spectively. T h e effect of t e m p e r a t u r e o n g r o w t h rate and e n z y m e p r o d u c t i o n was s t u d i e d . O p t i m u m g r o w t h t e m p e r a t u r e was 29°C., whereas o p t i m u m temperature for e n z y m e p r o d u c t i o n was 24°C. T h e m a x i m u m phosphate concentration not d e l e t e r i o u s to g r o w t h was 2 0 m M . 4

2

4

O t h e r methods of i n c r e a s i n g the specific heparinase p r o d u c t i o n of Flavobacteria c u r r e n t l y are u n d e r study. A strain i m p r o v e m e n t p r o g r a m of mutation a n d selection has b e e n i m p l e m e n t e d u s i n g ultraviolet and 7-irradiation, f o l l o w e d by g r o w t h selection methods. M a n y mutant cultures have b e e n o b t a i n e d u s i n g these m e t h o d s . O f those c u r r e n t l y u n d e r investi gation, one p a r t i c u l a r mutant has p r o v i d e d a twofold increase i n specific p r o d u c t i v i t y of heparinase over the w i l d type i n d e f i n e d m e d i u m . G e n e t i c m a n i p u l a t i o n studies w i l l be the m a i n focus o f c o n t i n u i n g work, w i t h the ultimate objective of o b t a i n i n g a constitutive mutant capable of p r o d u c i n g heparinase at h i g h levels.

31.

LANGER ET AL.


Purification of Heparinase.

499

T h e objectives of o u r w o r k o n the p u r i

fication of heparinase w e r e t w o f o l d : (1) to adapt previous p u r i f i c a t i o n schemes of H o v i n g h a n d L i n k e r (24) to large-scale p r o d u c t i o n of heparinase; a n d (2) to p u r i f y heparinase to h o m o g e n e i t y . T h e first goal has b e e n met largely b y m o v i n g f r o m a c o l u m n to a batch p u r i f i c a t i o n . T h e c e l l p e l l e t p r o d u c e d f r o m centrifugation of the f e r m e n t a t i o n b r o t h at 10,000 X g was r e s u s p e n d e d at 100 m g / m L p r o t e i n i n 0 . 0 1 M phosphate buffer at p H 7.0, and d i s r u p t e d sonically; the n u c l e i c acids w e r e p r e c i p i t a t e d w i t h 12.5 m g / m L of p r o t a m i n e sulfate; a n d the p r o t e i n solution was a d d e d to 4 g of hydroxylapatite p e r g of p r o t e i n . T h e h y d r o x y l a p a t i t e - b o u n d p r o t e i n was t h e n w a s h e d stepwise w i t h i n c r e a s i n g concentrations of N a C l a n d s o d i u m phosphate (from 0 . 0 M a n d 0 . 0 1 M to 0 . 5 0 M a n d 0 . 2 5 M , respectively). T h e r e s u l t i n g e n z y m e p r e p a ration ( H A ) , o b t a i n e d i n a 0 . 1 2 5 M N a C l a n d 0 . 0 7 M s o d i u m phosphate wash, was of sufficient p u r i t y to have its activity d e t e r m i n e d b y any of the available assays. A s a f u r t h e r p u r i f i c a t i o n t e c h n i q u e , affinity chromatography was ex p l o r e d . I n p r e l i m i n a r y e x p e r i m e n t s , a h e p a r i n - S e p h a r o s e c o l u m n failed to b i n d heparinase. W e therefore searched for a c o m p e t i t i v e and reversible heparinase i n h i b i t o r to act as a l i g a n d . T h r e e synthetic h e p a r i n s u b s t i t u t e s — p o l y v i n y l sulfate), p o l y a n e t h o l e sulfonate, and polystyrene sulfonate—met these r e q u i r e m e n t s .

The

i n h i b i t o r y effect o f p o l y v i n y l

sulfate) (Kj =

3.0 X 1 0 " M ; M W ~ 10,000) a p p e a r e d to be l i n k e d to the presence of sulfate 8

groups because i n h i b i t i o n was lost w h e n p o l y v i n y l sulfate) was h y d r o l y z e d .

Figure 3. Results of a typical fermentation on defined medium showing dry cell weight (Q), glucose ( Ό , heparin (A), and heparinase specific activity as a function of time in a 2-L fermentor (15j.

500

An


affinity c o l u m n was p r e p a r e d b y i m m o b i l i z i n g partially h y d r o l y z e d

p o l y v i n y l sulfate) o n epoxy-activated Sepharose (25). H e p a r i n a s e ( H A p u r i fied) was b o u n d to this c o l u m n , a n d was released at either h i g h or l o w p H (11 or 4) w i t h 5 - 1 0 % total activity recovery a n d u p to 5 0 0 % e n r i c h m e n t (21). I E F also was a p p l i e d towards the H A - p u r i f i e d e n z y m e to obtain h i g h l y p u r e heparinase. T h e e n z y m e was loaded onto a prefocused acrylamide g e l at p H 7.0. A f t e r I E F , the e n z y m a t i c activity was recovered at p H 8.5 ± 0.5. T h e r e s u l t i n g e n z y m e h a d a specific activity of about 5000 units/mg p r o t e i n , having u n d e r g o n e an e n r i c h m e n t of 50-fold (21). T h e p u r i f i c a t i o n o f heparinase has b e e n followed b y S D S gel electrophoresis. T h e c r u d e sonicate gave m o r e than 20 major bands; the H A p u r i f i e d e n z y m e , 3 major bands; a n d the I E F p u r i f i e d e n z y m e , 2 major bands. A s u m m a r y of the specific activities, p r o t e i n recoveries, a n d e n z y m e p u r i t y obtained u s i n g o u r p u r i f i c a t i o n p r o c e d u r e s is listed i n Table I. Properties of Heparinase.

O u r studies of the structure of heparinase

show it to have a m o l e c u l a r w e i g h t of 51,000 ± 6,000 b y Sephadex G - 2 0 0 gel exclusion c h r o m a t o g r a p h y , a n d 45,700 ± 1,600 w i t h o u t subunits b y S D S g e l electrophoresis. T h e e n z y m e is v e r y specific, acting o n l y o n h e p a r i n ( K m = 4.2 X 1 0 ~ M ) 5

and h e p a r i n monosulfate. H e p a r i n a s e acts e n d o l y t i c a l l y as an a - 1 , 4 - e l i m i nase cleaving h e p a r i n ( M W = 10,000) at 7 to 8 sites (42). D e t a i l e d studies have b e e n p e r f o r m e d o n the activity a n d stability of heparinase. H y d r o x y l a p a t i t e - p u r i f i e d heparinase ( H A ) is stable to f r e e z e thawing and f r e e z e - d r y i n g , w i t h 90 and 8 7 % recovered activity, respectively. H i g h l y p u r i f i e d heparinase r e q u i r e s the a d d i t i o n of B S A or p o l y l y s i n e (0.05%) a n d g l y c e r o l (7.5%) to p e r m i t 100% activity recovery o n f r e e z e thawing. T h e effect o f salts o n heparinase activity was e x a m i n e d . A n activity m a x i m u m was o b t a i n e d i n 0 . 1 6 2 M s o d i u m c h l o r i d e ; however, the m a x i m u m e n z y m e stability o c c u r r e d at a somewhat h i g h e r concentration. T h e effect of the cations C a

2 +

, Fe , Fe , Zn

Pb , L i , K , Hg 2 +

+

+

2 +

2 +

, Mg

3 +

2 +

, NH

2 +

4

+

, Cu

2 +

, Mo

, Al

3 +

, B a , was tested u s i n g the H A -

2 +

, Co

2 +

, Mn

2 +

, Sn , Cd

p u r i f i e d heparinase. Slight i n h i b i t i o n was shown b y B a , N H 2 +

but total loss of activity o c c u r r e d for H g

2 +

2 +

,

2 +

4

+

, and P b , +

at 1 0 " M .

2 +

5

T a b l e I. H e p a r i n a s e P u r i f i c a t i o n Procedure W h o l e cells Sonicate P r o t a m i n e precipitate Hydroxylapatite purified Affinity chromatography Isoelectric focusing

Specific Activity 4.3 7.9 12.5 88 2,000 5,000

0

Protein (mg) 1000 730 480 45 — —

"Milligrams of heparin degraded per milligram of protein per hour (15).

Major SDS Bands — 20 — 3 — 2

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501

Studies o f t h e effect o f p H o n H A heparinase activity a n d stability d e t e r m i n e d that the activity m a x i m u m occurs at p H 5.8, w h i l e the stability m a x i m u m occurs at p H 7.0. H A heparinase has an activity m a x i m u m at 30°C ( F i g u r e 4), b u t greater stability at l o w e r t e m p e r a t u r e ; f

1/2

d e n a t u r a t i o n at 4 ° C was 125 h a n d t

i/2

denaturation at 3 0 ° C was 25 h ( F i g u r e 5). Heparinase Immobilization.

H e p a r i n a s e has b e e n i m m o b i l i z e d o n a

variety of supports, w i t h a w i d e l y d i f f e r i n g degree of success. T h e best results have b e e n o b t a i n e d o n Sepharose a n d p o l y a c r y l a m i d e . L o w levels of activity recovery o c c u r r e d o n P H E M A . T h e other supports tested gave either no activity recovery o r o n l y b a r e l y detectable levels of activity (Table II). To check several o f the i m m o b i l i z a t i o n methods, chondroitinase A B C (from Proteus vulgaris) was u s e d as a c o n t r o l . A s u m m a r y of the activity recoveries of i m m o b i l i z e d heparinase a n d chondroitinase is listed i n Table I I .

TEMPERATURE ° C

Figure 4. Activity profile of heparinase. Key: Δ , specific activity of native enzyme, and Q > specific activity of the Sepharose-immobilized enzyme.

502


5000 -I

0

10

20

30

40

50

60

70

80

TEMPERATURE ( ° C )

Figure 5. Stability of heparinase. Key: A, half life of denaturation of native enzyme, and Q, half life of denaturation of Sepnarose-immobilized enzyme.

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Table II. Enzyme Immobilization on Various Polymer Supports Carrier

% Activity Immobilized Réf. Heparinase Chondroitinase

Coupled By

Sepharose Polyacrylamide

CNBr (26) (27) NHS active ester Polyacetal(aldehyde) (28) (28) Acyl hydrazide PHEMA (26,29) CNBr PHEMA(Spheron) (26,29) CNBr PMA-C0 H EDC (30) PMA-CONH(CH ) OH CNBr (26,30) PMA-BSA (CH ) (CHO) (30) PMA-{CH ) NH (CH ) (CHO) (30) PMA-(CH ) NH (CH ) (CHO) (30) Dacron (EtO) Si(CH ) N = CH(CH ) CHO (3D Dacron-NH (CH ) (CNO) (32) (CH ) (CHO) (33) Dacron-C0 H Woodwards Κ reagent (34) Silicone-NH (CH ) (CHO) (35) 2

2

2

2

2

2

2

6

2

2

6

2

2

6

2

2

6

3

2

2

2

3

2

2

6

2

2

6

2

2

6

2

3

2

2

0.8

91 56 1.2 0.7 1.6 0.03 0.01 0.0 0.0 0.0 0.0 0.2 0.0 1.0 0.2 0.0

— — — —

2.2 0.4

— — — —

10.0

— — —

0.4

T h e C N B r - a c t i v a t e d Sepharose 4 B support (1 g d r y weight) was s w e l l e d i n 25 m L o f h y d r o c h l o r i c a c i d ( 0 . 0 0 1 M ) , a n d t h e n washed w i t h 100 m L o f 0.5M NaCl, 0.1M N a H C 0

buffer at p H 8 . 3 . To this support 5 . 5 m L o f

3

h y d r o x y l a p a t i t e - p u r i f i e d heparinase (0.2 m g / m L p r o t e i n w i t h an activity of 88 units/mg p r o t e i n i n 0 . 2 M phosphate buffer at p H 7.0) a n d 60 m g o f h e p a r i n were a d d e d . T h e m i x t u r e was shaken overnight at 4°C., after w h i c h the beads were washed a n d b l o c k e d o v e r n i g h t at 4°C w i t h a solution of lysine at p H 8.2 in 0 . 5 M N a C l , 0 . 1 M N a H C 0

3

buffer s o l u t i o n . T h i s support showed an uptake

of 8 7 % of the p r o t e i n a n d an i m m o b i l i z a t i o n of 9 1 % of the heparinase activity. A t present, w e are c o n t i n u i n g o u r efforts to i m m o b i l i z e heparinase to support materials i n o r d e r to achieve h i g h e r y i e l d s . W h i l e this w o r k is i n progress, w e have b e g u n to e x p l o r e the properties of i m m o b i l i z e d heparinase using h e p a r i n a s e - S e p h a r o s e as a m o d e l . H e p a r i n a s e , i m m o b i l i z e d o n Sepharose, has e n h a n c e d t h e r m a l stability. T h i s effect is e s p e c i a l l y noticeable i n t h e l o w - t e m p e r a t u r e storage o f this e n z y m e . A t 4 ° C the i m m o b i l i z e d e n z y m e has a half life o f denaturation o f > 3600 h , c o m p a r e d w i t h a 125-h half life o f the native e n z y m e at the same temperature ( F i g u r e 5). T h e greater stability o f the i m m o b i l i z e d e n z y m e is also seen at h i g h e r t e m p e r a t u r e s : 25°C., f 60°C., t

l/2

1/2

= 1,000 h ; 37°C., ty = 15 h ; a n d 2

= 0.2 h .

A l o n g w i t h e n h a n c e d stability, t h e activity profile o f the e n z y m e is b r o a d e n e d over a larger t e m p e r a t u r e range as a result o f the i m m o b i l i z a t i o n . T h e activity m a x i m u m is shifted to a slightly h i g h e r temperature, f r o m Τ = 30°C for t h e free e n z y m e to Τ = 37°C for t h e S e p h a r o s e - i m m o b i l i z e d e n z y m e ( F i g u r e 4). T h i s result m a y reflect the temperature d e p e n d e n c e o n

504


the rate of substrate diffusion into the support. T h e p H m a x i m u m of b o t h the native a n d i m m o b i l i z e d (on Sepharose) e n z y m e are i d e n t i c a l (data n o t shown). T h e apparent K

m

of the i m m o b i l i z e d e n z y m e is 1.2 X 1 0 ~ M (this can b e 3

d e t e r m i n e d b y r e p l o t t i n g the data i n F i g u r e 6 on a L i n e w e a v e r - B u r k plot). T h i s K is c o n s i d e r a b l y h i g h e r than t h e K d e t e r m i n e d for the free e n z y m e m

(K = 4 . 2 x m

m

10" M). 5

I n V i t r o Studies o n I m m o b i l i z e d H e p a r i n a s e .

I n i t i a l experiments have

b e e n c o n d u c t e d to test t h e effectiveness o f i m m o b i l i z e d heparinase i n r e m o v i n g h°parin i n v i t r o . C o n t r o l s consisted o f Sepharose-heparinase

that

was d e n a t u r e d b y h e a t i n g at 100°C for 3 0 m i n . I n o n e set o f experiments, both active a n d d e n a t u r e d i m m o b i l i z e d heparinase w e r e loaded into two c o l u m n s , b o t h w i t h a 1 . 5 - m L b e d v o l u m e . Solutions o f h e p a r i n , B S A (60

1.0

Feed Concentration (mg/ml) Figure 6. The conversion of heparin to products as the result of passing a sodium acetate buffer (pH 7.0, 0.25M) containing heparin through a 1.5-mL Sepharose-heparinase column. The conversion was measured as both a function of feed concentration (x-axis) and flow rate (mL/min). Key: 0.1; A, 0.2; Q, 0.3;O, 0.4; V , 0.5.

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mg/mL), a n d salts w e r e passed t h r o u g h each c o l u m n at a flow rate o f 0.5 m L / m i n . T h e concentrations of the n o n h e p a r i n species w e r e chosen to m i m i c physiological concentrations. T h e h e p a r i n levels i n the solution increased i n stepwise fashion f r o m 15 μg/mL to 75 μg/mL, as shown i n F i g u r e 7. A s t h e h e p a r i n l e v e l i n the i n p u t solutions increased, the difference i n the h e p a r i n r e c o v e r e d at t h e outlet o f t h e c o n t r o l a n d active c o l u m n s also increased ( F i g u r e 7). E v e n at 66 μg/mL the h e p a r i n was largely r e m o v e d b y the active 70

Heparin Feed

60 50 40 30

I

20

r

10 0

Figure 7. Heparin removal from a protein!salt solution by a 1.5-mL column packed with Sepharose-immobilized heparinase. The upper portion represents the stepped increase of heparin input; the bottom portion measures heparin output from both the Sepharose-immobilized enzyme (A) and the heat-denatured Sepharose-immobilized enzyme (Q).

506


heparinase c o l u m n , w h i l e the d e n a t u r e d heparinase c o l u m n had no effect. C l i n i c a l l y used levels of h e p a r i n are o n the o r d e r of 5 - 1 0

μ^πιΕ.

In a second e x p e r i m e n t , the effect of b o t h h e p a r i n concentration and flow rate o n h e p a r i n degradation was e x a m i n e d . T h e same size c o l u m n as was used i n the p r e v i o u s e x p e r i m e n t was e m p l o y e d . A s shown i n F i g u r e 6, at l o w flow rates this s m a l l c o l u m n was fully capable of d e g r a d i n g v e r y large q u a n tities of h e p a r i n (more than 100-fold i n excess of c l i n i c a l l y used amounts) i n a single pass. W e have just b e g u n a series of experiments i n w h i c h citrated rabbit b l o o d , h e p a r i n i z e d at a l e v e l of 10 u n i t s / m L (153 units/mg), was passed through a S e p h a r o s e - h e p a r i n a s e c o l u m n (0.5mL) at a flow rate of 0.5 m L / m i n ( F i g u r e 8). A f t e r 5 m i n the b l o o d l e a v i n g the b o t t o m of the c o l u m n was s a m p l e d a n d assayed for h e p a r i n b y w h o l e b l o o d c l o t t i n g t i m e and Factor Xa h e p a r i n assays. I n the active c o l u m n , 5 0 % of the h e p a r i n was r e m o v e d . H o w e v e r , w h e n the same h e p a r i n i z e d b l o o d was treated w i t h a c o n t r o l c o l u m n , less than a 5 % decrease i n anticoagulant activity was observed.

Discussion T h e s e studies p r o v i d e i n i t i a l data for d e v e l o p i n g a system to remove h e p a r i n i n extracorporeal therapy. Because the amount of data o n heparinase has b e e n l i m i t e d , u n t i l n o w , and the methods of p r o d u c i n g it inadequate for large scale use (24), the focus of o u r research thus far has b e e n on d e v e l o p i n g the necessary technology for e n z y m e p r o d u c t i o n and p u r i f i c a t i o n . T h e p r i n cipal c o n t r i b u t i o n s of o u r studies have b e e n (1) increasing p r o d u c t i o n levels of heparinase b y over 1000-fold (15) f r o m p r e v i o u s l y p u b l i s h e d p r o c e d u r e s (24), (2) p u r i f y i n g heparinase by over 1000-fold f r o m the c r u d e c e l l extracts, (3) c h a r a c t e r i z i n g the p r o p e r t i e s of heparinase and isolating the first hepa rinase i n h i b i t o r s , (4) i m m o b i l i z i n g the e n z y m e w i t h 9 1 % activity recovery and excellent stability, a n d (5) d e m o n s t r a t i n g that c o l u m n s as small as 1.5 m L can remove c l i n i c a l l y u s e d quantities of h e p a r i n i n aqueous m e d i u m and i n blood. T h e d e v e l o p m e n t of the h e p a r i n r e m o v a l system is still at an early stage. W o r k c u r r e n t l y is b e i n g d i r e c t e d toward (1) c o m p l e t i n g the p u r i f i c a t i o n of heparinase, (2) i m m o b i l i z i n g heparinase to a d d i t i o n a l supports, a n d (3) test i n g the b l o o d c o m p a t i b i l i t y a n d effectiveness of heparinase reactors i n v i t r o and i n vivo. O n e of the c r i t i c a l factors i n o u r research has b e e n the adaptation and use of m u l t i p l e assays to f o l l o w heparinase activity. Particularly i m p o r t a n t were assays (e.g., A z u r e A) used i n m o n i t o r i n g the fermentation and early stages of p u r i f i c a t i o n . B y u t i l i z i n g three different approaches for assaying h e p a r i n (disappearance of h e p a r i n , appearance of reaction products, and disappearance of heparin's b i o l o g i c a l activity), the occurrence of any arti-

31.

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507

facts i n the p r o d u c t i o n a n d p u r i f i c a t i o n p r o c e d u r e s , a n d activity tests, was avoided. W h i l e o u r studies o n heparinase p r o d u c t i o n and p u r i f i c a t i o n have b e e n encouraging, less success has b e e n a c h i e v e d i n t h e i m m o b i l i z a t i o n p r o cedures (Table II). Studies are i n progress to u n d e r s t a n d better the i m p o r t a n t parameters i n i m m o b i l i z a t i o n p r o c e d u r e s a n d i n establishing n e w supports. Initial results i n d i c a t e that a n o n c h a r g e d support w i t h a h i g h surface area is best (Table II). A d d i t i o n a l l y , o u r p r e l i m i n a r y evidence is that h i g h levels o f heparinase ( > 1 mg/mL) a n d the presence of substrate i n the i m m o b i l i z a t i o n reaction enhance t h e r e c o v e r y o f i m m o b i l i z e d e n z y m e activity.

ioq

> u < H Ζ

Ο < Ο υ

ζ

A System for Heparin Removal - Advances in Chemistry (ACS

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