Extracorporeal removal of specific antibodies by hemoperfusion through the immunosorbent...

Extracorporeal removal of specific antibodies by hemoperfusion through the immunosorbent agarose-polyacrolein microsphere beads: Removal of anti-bovine serum albumin in animals

Leon Marcus Department of Plastics Research, The Weizmann Institute of Science, Rehovot, Israel, 76100 Aharon Mashiah Department of Vascular Surgery, Kaplan Hospital, Rehovot, Israel Michael Offarim and Shlomo Margel* Department of Plastics Research, Weizmann Institute of Science, Rehovot, Israel, 76100

We describe (1) the production of a unique immunosorbent system, agarose-polyacrolein microsphere beads (APAMB) for removal of a specific antibody, anti BSA, and (2) its efficacy in animal trials. This is a model system for hemoperfusive removal of specific antibodies or antigens directly from whole blood. The agarose beads (1.0 mm mean diameter) contain thousands of microspheres of 0.2 pm mean diameter. The microspheres which contain the ligand are encapsulated within an agarose matrix to confer physical strength, biocompatibility, spacial configuration, and porosity allowing rapid entry of plasma for reaction. Any antigen may be linked covalently to spacers on the polyacrolein microspheres to remove a specific antibody, or vice

versa. Thus the APAMB remove specific molecules in contrast to the charcoal or ion exchange resins currently in use. Removal of antibody is efficient and rapid, there- fore, short hemoperfusive times may be used. The beads are biocompatible; there are negligible decreases in RBC, WBC and platelets. Electrolytes and other soluble components also are minimally affected. Therapy, at the least palliative, of autoimmune disorders i.e., multiple myeloma, macroglobinemia, autohemolytic anemias, idiopathic thrombocytopenia, myasthenia gravis, rheumatoid arthritis, thyroiditis, glomerulonephritis, etc, is potentially available with this or its further improved versions.

INTRODUCTION

Extracorporeal procedures (continuous flow plasma/cell separation, hemodialysis and hemoperfusion) are commonplace in clinical medicine. However, hemoperfusion appears to be the method of choice for direct detoxification of blood. State-of-the-art extracorporeal hardware is available and is in current use in hemodialysis. The next breakthrough in hemoperfusion must concern adsorbents. Activated charcoal' and ion exchange resins2f13 are the principal sorbents commercially available to remove toxic

*Address reprint requests to Dr. Shlomo Margel, Dept. of Plastics Research, The Weizmann Institute of Science, Rehovot, Israel, 76100. Journal of Biomedical Materials Research, Vol. 18, 1153-1167 (1984) 0 1984 John Wiley & Sons, Inc. CCC 0021-9304/84/091153-15$04.00

1154 MARGEL ET AL.

molecules directly from the blood stream. Both are biocompatible following encaps~lation.~-~ These adsorbents have been employed for medical emer- gencies: hepatic failure,8 renal f a i l ~ r e , ~ drug self iatrogenic overdose,ll and metal poisoning.12 Although dozens of other uses limited only by the imagination of the investigator-clinician have been tried or suggested; see reviews. 13-16 Unfortunately charcoal and ion exchange resins are non- specific and remove physiologically desirable biocompounds indiscrimi- nately in addition to toxic substances. For example, compounds listed in a catalog of molecules removed by charcoal or resin hemoperfusion include amino acids, polypeptides, and polypeptide hormones, ”middle molecules,” thyroxine, triiodothyronine, epinephrine, norepinephrine, L-dopa- mine, serotonine, nucleotides, vitamin B12, and folic acid. l3

We have designed and produced microspheres to which a wide variety of proteinaceous and other molecules may be bound c~valen t ly . ’~-~~ Thus medically useful microspheres with desginated properties are potentially available. Thus far, polymercaptal microspheres for removal of mercury from blood and other fluids have been produced and currently are under- going biomedical trials.20,21 Immunospheres with specific antibodies on their surface have been used for labelling and separating T from B lympho- cyte~.~’-’~ In addition, magnetic properties can be conferred on the microspheres so that the cells can be labeled and separated in a magnetic field.24

We report herein a specific absorbent system: Agarose-PoIyacrolein Mi- crosphere Beads (APAMB). These are agarose beads of 1.0 mm diameter which contain thousands of polyacrolein microspheres of 0.2 pm mean diameter, encapsulated in an agarose matrix. A specific antigen is covalently linked to spacers on the polyacrolein microspheres to remove a specific antibody or vice versa. The gel matrix coating confers physical strength, biocompatibility, and spatial configuration as well as porosity to permit rapid entry of antibodies for reaction. Specific APAMB should be useful for therapy or palliation of syndromes secondary to immunological disorders.

In our model system the ligand on the microspheres is bovine serum albumin (BSA),25 although in practice any molecule containing primary amino groups may be covalently bound to the microspheres through their aldehyde groups. Circulating anti-BSA antibodies bind to the microspheres via an antigen-antibody reaction. Subsequently, antibody may be eluted quantitatively from the beads.

Previously, several in vivo systems for elimination of specific antigens or antibodies have been described. In one, blood flows through a tube coated with entrapped immunosorbent.26 In another,27 antigen is bound to nylon tubing, which itself is passed into the vascular bed to remove antibody during circulation. In a third, sorbent is encapsulated and placed into a column. 5,28

A less specific but very exciting approach seems to be clinically success- fully, that is using (1) Protein A bound to charcoal colloidion for therapy of solid tumors” and (2) a heat killed, stabilized S. aureus sorbent to decrease serum IgG in an autoimmune hemolytic anemia syndrome.30

IN VIVO HEMOPERFUSIVE REMOVAL OF ANTIBODIES 1155

This new immunoadsorbent system we report is capable of removing specific antibodies from the bloodstream, leaving the other blood components intact. This is our model system presaging an attack on several autoimmune disorders.

MATERIALS AND METHODS

Preparation of the agarose-polyacrolein microsphere beads (APAMB)

Synthesis of the polyacrolein microspheres.

Acrolein is added to a 0.5% (wh) solution of the surfactant polyethylene oxide to a final concentration of 10% (w/v). The mixture is deaerated with argon and irradiated with a 1 Mrad cobalt source. The resultant microspheres, 0.2 km mean diameter, are dialyzed against water and centrifuged through distilled water four times at 1800 g for 10 min.

Production of the APAMB

The procedure for the production of matrix-encapsulated micro sphere^'^,^^ is similar to that of Losgen et al.31 A solution of 0.8 g agarose in 12.5 mL deionized water is heated to 95°C to melt the gel into a clear solution and then held at 70°C. Aqueous polyacrolein microspheres, 12.5 mL, (10% w/v) at 70°C is added to make a stirred suspension in agarose and is drawn into the glass syringe apparatus described previously.20 The syringe apparatus is maintained at 70°C while the suspension is stirred at 300 r.p.m. The gel is expressed via the motor driven piston dropwise into a tall vessel which contains an ice-cold solvent mixture of toluene, chloroform, and hexane in the ratio 10:4:2. The beads formed are separated from the solvent mixture in a coarse sieve, washed several times with dioxane, and finally cleared of solvents by exhaustive washing with deionized water. The APAMB are stored at 4°C in 0.05% sodium azide.

Binding spacers to the APAMB18

Twenty milliliters of the APAMB in 35 mL distilled water are shaken for 24 h with 100 mg of polylysine. The resultant polylysine conjugated beads are freed from unreacted polylysine by repeated decantation from water. The bead suspension (20 mL in 35 mL water) is shaken for 12 h with 2 mL of 50% glutaraldehyde and the resultant polylysine-glutaraldehyde conjugated beads are filtered and then washed with copious quantities of distilled water.

Cross-linking the APAMB

One milliliter APAMB was added to an aqueous solution containing 2 mL of 0.5 M sodium carbonate buffer (pH 11.0) and 10 FL divinyl sulfone (DVS).

1156 MARGEL ET AL.

The cross-linking reaction was allowed to continue for 2 h at 40°C. The cross-linked APAMB were subsequently freed of DVS with numerous washings through phosphate-saline buffer (PBS), pH 7.4. The remaining unreacted double bonds of the DVS were quenched with ethanol for 2 h at 40°C. The ethanol was removed with numerous washings through PBS. Beads formed in this manner are stronger; more resistant to agglomeration and shattering and are sterilizable by autoclaving.

Preparation of the irnmuno~orbents~~~~~

Twenty milliliters of APAMB with bound spacer are shaken for 24 h at 4°C with 400 mg of an appropriate antigen, in this case BSA, in 200 mL PBS. Unbound antigen is removed by repeated decantation with large amounts of PBS. Remaining aldehyde groups are then blocked by shaking the beads with 1 mL of aqueous ethanolamine (pH 7.2) for 12 h. The immunobeads are then washed successively with PBS, eluting medium (0.2 M glycine HC1, pH 2.4) and equilibrated with PBS.

Elution of the bound a n t i b ~ d y l ~ , ~ ~

After hemoperfusion the immunobeads are rinsed free of residual blood with PBS until the absorbancy of the effluent at 0.80 pm falls below 0.02. Antibodies are then eluted with 0.2 M glycine HC1, pH 2.4 and returned to neutral pH before quantification. The immunobeads likewise are returned to pH 7.4 with PBS and stored at 4°C in PBS-0.05% Na azide if they are to be reused. In our hands the APAMB-BSA are stable; they have been used in hemoperfusion trials, stripped of sorbed molecules, reused tens of times and still retain their capacity after 15 months.

Capacity of the APAMB

The capacity of the APAMB was determined by perfusing immune rabbit serum through the beads until maximal practical uptake of the anti BSA occurred; the content of the anti BSA exceeded the adsorptive capacity of the beads. The anti BSA level in the serum remained constant. Adsorbed anti BSA was eluted from the column; the amount of anti BSA remaining in the serum as well as the amount of anti BSA adsorbed was quantitated by the RIA m e t h ~ d . ' ~ , ~ ~

Specificity of the APAMB-BSA system.

and subjected to elution according to the standard p r o c e d ~ r e . ' ~ , ~ ~ Normal rabbit serum perfused the APAMB-BSA; the beads were rinsed


Animals

Inbred white rabbits were obtained from the Dept. of Experimental Ani- mals of the Weizmann Institute of Science.

Immunization

Rabbits weighing 3.5 to 4.5 kg were immunized with an emulsion of BSA in Freundts complete adjuvant (1 mg/mL BSA, final concentration). Each rabbit received 2 mL injected intradermally into at least eight different sites. The rabbits were boostered monthly after the first dose in the same manner, as required.

ZN VIVO EXTRACORPOREAL SYSTEM

Anesthesia

In early trials, rabbits were anesthetized with ether via a nose cone. More recently, Ketamine HCl (Parke Davis), 15 mgkg was injected IM and minimal additional ether was required. Oxygen was administered via a face mask, if needed.

Cannulization

Cannulas are ordinary umbilical lines, French 8 (Portex) which are cut to length and inserted into the carotid artery and jugular vein. After place- ment, the cannulas were passed under the skin and externalized at the back of the neck.32

Hemoperfusion tubing and pumping system

The in situ cannulas were connected to a specially designed hemoperfusion arterial outlet set via a three-way stopcock. Arterial blood flowed from the carotid artery through the pump segment sited in the peristaltic pump (Watson Marlow, Ltd., MHRE 200, Falmouth, Cornwall, England), to per- fuse through the APAMB in the column. Inline Tee connections permit continuous blood pressure monitoring and precolumn infusion as needed. The other side of the column was attached to the venous inflow set, con- sisting of a bubble trap, Tee connection and tubing which end in a three- way stopcock. An inline Tee fitting admits IV infusion as needed. Blood returns to the rabbit via the stopcock to the cannula in the jugular vein.32

Column

The perspex column consists of a cylinder, diameter, and length according to need, with standard outer threading at both ends. The column screws

1158 MARGEL ET AL.

into identical end pieces gently tapered at their inner distal ends, fitted with Teflon connectors. The APAMB were retained by 40 mesh stainless-steel screening fitted into a Teflon holder.32

All plastic components are medical grade-biocompatible and were steril- ized with ethylene oxide prior to use.

The complete system including tubing, APAMB, bubble trap, etc., were equilibrated with sterile heparinized saline (10 U/mL) and flushed with heparinized saline (1 U/mL) before use. The extracorporeal blood was temper- ature controlled in a water bath at 39°C (Fig. 1).

Whole blood perfused the APAMB-BSA at speeds of 8 to 15 mL per min so that the entire blood volume perfused the APAMB every 20 to 30 min.

Monitoring blood pressure

Blood pressure was monitored intermittently via tubing connected to the three-way stopcock immediately at the arterial outflow with the transducer assembly and monitor manufactured by the Mennen Electronics Co., Kiryat Weizmann, Rehovot, Israel. Newer tubing sets contain an additional Tee connector close to the arterial blood out flow for continual arterial blood pressure measurements.

Heparination

Rabbits were fully heparinized by IV injection of 300 U heparidkg (Or-


ganon) and maintained anticoagulated by heparin drip or discrete addition as needed according to clotting times determined during the course of hemoperfusion.

The rabbits were lightly restrained though unanesthetized during hemoperfusion. They seemed quite content and usually dozed during the procedure.

Assays

uble components and antibodies. Blood was sampled at intervals for quantitation of formed elements, sol-

Enumeration of the formed elements of the blood

Formed elements of the blood were counted manually or with a Coulter Counter in the Hematology Dept of Beilinson Hospital, Petah Tikvah, Israel.

Soluble components of the blood

The battery of soluble components of the serum was quantified with a Technicon SMA Autoanalyser in the SMA Chemistry Lab of Beilinson Hos- pital.

Antibody assay

Antibody content was determined in triplicate with the RIA technique.33

RESULTS

Photomicrography

Figure 2(a) is a photomicrograph of the APAMB; mean diameter of the beads is 1.0 mm. Figure 2b is a cross sectional photomicrograph of the APAMB showing the microspheres encapsulated within the agarose matrix; mean diameter of the microspheres is 0.2 pm.

Capacity of the APAMB

gm APAMB.25 The capacity of this batch of APAMB was 10 mg anti-BSA adsorbed per

Specificity of the APAMB-BSA system

Following in vitro perfusion of normal rabbit serum through the APAMB- BSA and rinsing, no protein was eluted from the beads. Thus it is shown that the APAMB do not adsorb proteins nonspecifically. The APAMB can bind cross reactive anti BSA from other genera, but such conditions do not

1160 MARGEL ET AL.

Figure 2. (A) Photomicrograph of the APAMB (original magnification: 18 x .) (B) Thin section through the APAMB showing the encapsulated microspheres. Partial circumferences of two beads are shown (original magnification: 4,000 X .)

apply in these hemoperfusions. In these trials, antibodies were produced and removed from rabbits. When the eluted anti-BSA were subjected to polyacrylamide gel electrophoresis, only one band of IgG was detected.

Rate of removal of anti-BSA from immune serum (in vitro)

Column overload condition

Pooled serum from immune rabbits was perfused through the APAMB in the standard procedure. Figure 3 shows the rate of removal of anti-BSA from the serum. 50% of the anti-BSA was removed in 30 min; 62% in 60 min; 73% in 120 min, and 79% in 180 min.

Column operated at less than capacity

The companion experiment to the above in which the rabbit anti-BSA was well within the capacity of the APAMB is described in Figure 4. Sixty-two


u O"0 30 60 90 120 I50 180

MIN9TES

Figure 3. Rate of removal of anti-BSA from immune rabbit serum: Column overload condition. Column overload condition. Column contained 15 g APAMB. Resevoir contained 100 mL serum. Pump speed = 15 mWmin.

percent of the anti-BSA was removed in 30 min; 78% in 60 min; 91% in 120 min, and more than 95% in 180 min. In additional experiments, over 90% of the anti-BSA was removed in 120 min and almost quantitative removal occurred within 180 min.

Rate of removal of Anti-BSA from immunized rabbits (in vivo)

Column overload condition

Immunized rabbits were fitted with cannulas and coupled to the extracorporeal system. A 3.5-kg rabbit containing 147 mL serum (calculated); 3.6 mg anti-BSA per mL serum (529 mg anti-BSA total) was hemoperfused. The case where the APAMB were grossly overloaded is shown in Figure 5. Forty- four percent of the anti-BSA was removed in 30 min; 51% in 60 min, and the APAMB were saturated within 105 min. Three hundred twenty three

M I N'J TES Figure 4. Rate of removal of anti-BSA from immune Rabbit serums: Within column capacity. Column contained 15 g APAMB. Resevoir contained 100 mL serum. Pump speed = 15 mL/min.

1162 MARGEL ET AL.

2 2 5

6 2 0 tr m

15 [L

u 0 60 120 180

M I N U T E S

Figure 5. Rate of removal of anti-BSA from immunized rabbit: column overload condition. Rabbit weighing 3.5 kg with 3.6 mg anti-BSA per mL plasma. Upper and lower limits include data for three rabbit hemoperfusions normalized to this rabbit. Column contained 30 g APAMB. Pump speed = 12 mL/min.

milligrams of anti-BSA was removed. Upper and lower values for three rabbits are given in the figure.

Column operated within capacity

A 3.9-kg rabbit containing 0.55 mg anti-BSA per mL serum (total of 90 mg anti-BSA) was hemoperfused; the results are shown in Figure 6. Sixty-two percent of the anti-BSA was removed in 30 min; 78% in 60 min; 95% in 120 min, and 96% in 180 min. Upper and lower values for four hemoperfusions are presented in the figure.

Biocompatibility of the APAMB

Soluble components of the blood

Serial blood samples were drawn during the course of hemoperfusion and the routinely useful soluble components examined. Results are tabulated in Table I. There was little variation in the ionic species, urea, uric acid, or creatinine.

Formed elements of the blood

Blood was sampled serially simultaneously with the above and the cells enumerated. Table I shows the counts. After 3 h the RBC count declined by 2%; the WBC count by 4%) and thrombocytes by 4%.

Upper and lower limits for formed elements and soluble components of the blood for seven rabbit hemoperfusions fell between ?3% of the means presented.


06

m 2 0 3 0

m

0 60 120 180 M I N U T E S

Figure 6. Rate of removal of anti-BSA from immunized rabbit: within column capacity. Rabbit weighing 3.5 kg with 0.55 mg anti-BSA per mL plasma. Upper and lower limits include data for four rabbit hemoperfusions normalized to this rabbit. Column contained 30 g APAMB. Pump speed = 12 mL/min.

DISCUSSION

Described herein is the development and use of biocompatible adsorbents to remove specific antibodies directly from the bloodstream. Anti-BSA antibodies are removed efficiently at a rate practical for hemoperfusion.

The microspheres used in these studies are encapsulated in a matrix of agarose, a standard coating which confers biocompatibility. Using APAMB to which 1311 BSA or DNP BSA was bound, we have not detected leaching of antigen from the microspheres, shattering of the beads nor loss of microspheres from the beads during the lifetime of the I3lI. After 3 months no radioactivity, protein, or covalently bound DNP leaked from the beads. En- capsulation in agarose permits free permeability to macromolecules for in- teraction with more than adequate blood flow rates.7 Cross-linking the beads confers greater physical strength, i.e., the APAMB are more resistant to agglutination and shattering and also withstand autoclaving for steriliza- tion.

The lability of the formed elements of the blood is a continuing problem plaguing all extracorporeal techniques. In these trials the effect on the blood cells is minimal. We see decreases in the WBC and platelet final counts of 4%; sometimes slightly higher in the 60- and 120-min counts. Apparently the WBC and thrombocytes attach to the sorbent (in this and other systems). The counts tend to rise toward the initial values during the later stages of hemoperfusion as the cells detach from the column. It may be possible to eliminate the latter decreases by infusing citrate into the blood just prior to entry into the column.34 We have not tried this technique.

Of the soluble blood components, Cl-, K', Na', Ca2+, PO:-, urea, uric acid, and creatinine, were unaffected by the hemoperfusion. Likewise total protein and albumin remained fairly constant during the procedure. K+, PO:+, albumin and total protein in our untreated rabbits at zero time were usually significantly lower than the values recorded in a standard text3' although other texts report wide ~ a r i a t i o n . ~ ~

1164 MARGEL ET AL.

TABLE I Blood Constituents of a 3.9-kg Rabbit during Hemoperfusion'

Column Contained 30 g APAMB

Soluble Component Time (min)

0 60 120 180 C1- (mEq/L) 110 104 115 109 K+ (mEq/L) 3.2 2.7 2.0 3.3 Na' (mEq/L) 142 144 142 146 Ca2+ (mg%) 13.0 14.2 11.3 11.6 PO:- (mg%) 2.9 2.7 2.6 3.0 Urea (mg%) 18.0 16.0 14.0 14.0 Uric acid (mg%) 1.3 1.4 1.7 1.6 Creatinine (mg%) 1.3 1.6 1.5 1.4 Albumin (gm%) 3.1 2.9 2.7 2.5

Cell counts of the formed elements Time (min) RBC Hct WBC Thrombocytes

'ITL Protein (gm%) 6.3 6.1 5.5 5.3

0 4,800,000 30.6 6,000 233,000

120 4,500,000 28.8 5,300 230,000 60 4,500,000 28.7 5,600 220,000

180 4,700,000 29.9 5,700 220,000

a Initial blood values of a total of seven rabbits were normalized to those shown for this

Note. Blood pump speed = 12 mL/min. rabbit. All data fell within 2 3% of the mean values shown.

We do not foresee reuse of APAMB under clinical conditions. However, the adsorbed antibodies easily may be eluted. Thus, if the adsorbate is desired for further study it is readily obtainable.

Our system offers several advantages over those previously reported: (1) in the formation of the APAMB-BSA the protein is bound in a single step under physiological pH, thereby protecting sensitive proteins from denatur- ation, (2) it is specific, (3) whole blood may be perfused directly over the APAMB, (4) the reactive components of the beads are accessible to high diffusion rates, (5) it does not affect the formed elements of the blood, (6) additives to the blood are not required and (7) it has a high capacity allowing relatively small columns and minimal extracorporeal blood.

The importance of this work is that autoimmune syndromes, e.g., myasthenia gravis, rheumatoid arthritis, thyroiditis, glomerulonephritis, etc., which seem to be treatable with plasmapheresis are now potentially ame- nable to more specific therapy. Instead of removing all serum components; subsequently reinfusing serum albumin or fresh frozen plasma, with this system only specific antigens, antibodies or antibody complexes need be removed.

We thank Eitan Eshel, Migada Co, Kiryat Weizmann, Rehovot, Israel, for counsel during the designing and fabrication of several prototypes of cannulas and hemoperfusion outflow and inflow sets and for providing our laboratory with many sets for evaluation until the current models; the SMA Autoanalyser Laboratory of Beilinson Hos-


pital, Petah Tikvah, Israel for analyses of blood samples; and Michael Dalit of the Institute of Physiology and Experimental Surgery of the Beilinson Medical Center, Petach Tikvah, for discussions concerning animal physiology,

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Received May 1, 1984 Accepted June 26, 1984

Extracorporeal removal of specific antibodies by hemoperfusion through the immunosorbent...

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