Development of Affinity Microparticles for ExtracorporealBlood Purification Based on Crystalline Bacterial Cell

Surface Proteins

*Viktoria Weber, †Stefan Weigert, †Margit Sára, †Uwe B. Sleytr, and*Dieter Falkenhagen

*Christian Doppler Laboratory for Specific Adsorption Technologies in Medicine, Centre for Biomedical Technology,Donau-University Krems, Krems; and †Centre for Ultrastructure Research and Ludwig Boltzmann-Institute For

Molecular Nanotechnology, University for Agricultural Sciences, Vienna, Austria

Abstract: In this article, the development of specific ad-sorbents for extracorporeal blood purification are de-scribed. Affinity microparticles were prepared by linkingProtein A to crystalline cell surface layers (S-layers) fromThermoanaerobacter thermohydrosulfuricus l111-69. S-layers were used in the form of cell wall fragments ob-tained by breaking whole cells by ultrasonification, result-ing in cup-shaped structures (average size 0.5 × 1�m)completely covered with S-layer protein. Protein A wascovalently bound to carboxylic acid groups of the S-layerprotein after activation with 1-ethyl-3,3’(dimethylamino)-propylcarbodiimide. In batch adsorption experiments with

fresh frozen human plasma, the resulting S-layer basedaffinity microparticles showed a high adsorption capacityfor IgG (40 mg IgG were bound per g wet pellet of S-layerbased affinity microparticles). Fractions eluted from themicroparticles were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They contained onlyIgG demonstrating that adsorption was specific. In bio-compatibility tests, preparations of the S-layer micropar-ticles showed no low-density lipoprotein-reactivity, no cy-totoxicity, and no cytokine inducing activity. Key Words:Adsorption—Microparticles—Immunoglobulins—Bloodpurification—Biocompatibility.

Currently, a variety of extracorporeal proceduresfor therapeutic immunomodulation are available (1).These include plasma exchange, double filtration,immunoadsorption by means of immobilized ligandssuch as Protein A, DNA, C1q, antibodies or aminoacids, and chemosorption onto dextran sulfate. Themechanisms underlying extracorporeal immuno-modulation comprise the removal of pathogenicantibodies and circulating immune complexes(CIC). Furthermore, extracorporeal techniquesgreatly influence the stoichiometry, the structure,and hence the pathogenicity of CIC. Complementactivation during the passage of plasma through theextracorporeal circuit can prevent the precipitationof CIC or solubilize precipitated CIC (2). Beyondthat, the removal of CIC also results in deblockage

of the reticuloendothelial system of the liver andspleen.

Clinical applications of immunoadsorption includethe treatment of autoimmune diseases such as sys-temic lupus erythematosus, myasthenia gravis, sys-temic vasculitis, and rheumatoid arthritis (3–6).Moreover, immunoadsorption has been successfullyapplied for the removal of anti-human leukocyte an-tigen (HLA) antibodies as an adjunctive measure toavoid graft rejection in transplanted patients as wellas for the removal of factor VIII antibodies (7). Con-ventional immunoadsorption techniques are basedon the perfusion of separated plasma through ad-sorption columns and reinfusion of the purifiedplasma together with the concentrated blood cells. Inan alternative approach realized in the microspheresbased detoxification system (MDS), the plasma fil-trate does not perfuse an adsorption column, but isrecirculated into the filtrate compartment of themodule. The addition of a suspension of micro-spherical particles to the circulation system allows

Received April 2001.Address correspondence and reprint requests to Dr. Viktoria

Weber, Centre for Biomedical Technology, Donau-UniversityKrems, Krems, Austria. E-mail: weber@donau-uni.ac.at

Therapeutic Apheresis5(5):433–438, Blackwell Science, Inc.© 2001 International Society for Apheresis

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the rapid and, depending on the adsorbent, also thespecific adsorption of pathogenic substances at highcapacity (8,9).

Crystalline cell surface layers (S-layers) composedof identical protein or glycoprotein subunits havebeen identified as the outermost cell envelope com-ponents of many bacteria and archaea (10). High-resolution electron microscopic studies revealed thatS-layers exhibit oblique, trigonal, square, or hexago-nal symmetry. Their repetitive physicochemicalproperties down to the subnanometer range andtheir high density of functional groups make themparticularly suitable as matrices for the controlledimmobilization of macromolecules (11–13). In prin-ciple, there are 2 ways to apply S-layers as matricesfor adsorption purposes: cell wall fragments (averagesize: 0.5 × 1 �m) consisting of the rigid peptidogly-can-containing cell wall layer completely coveredwith S-layer subunits may be employed as affinitymicroparticles after covalent attachment of antibod-ies or synthetic peptides. Alternatively, micropar-ticles consisting of biocompatible polymers (e.g., cel-lulose microbeads) may be coated with purifiedS-layer proteins resulting in matrices with regularand defined surface properties for the immobiliza-tion of affinity ligands. Both affinity microparticlesand microbeads coated with S-layer protein are suit-able for use in the MDS-technology.

MATERIALS AND METHODS

ChemicalsProtein A from Staphylococcus aureus was pur-

chased from Amersham Pharmacia Biotech(Uppsala, Sweden). Glutaraldehyde was obtainedfrom Fluka (Buchs, Switzerland). All other chemi-cals were from Sigma (St. Louis, MO, U.S.A.). Freshfrozen human plasma (FFP) was obtained from thelocal hospital.

Bacterial strain and growth conditionsT. thermohydrosulfuricus L111-69 was grown in

continuous culture on S medium under strictly an-aerobic conditions in a 10 L Biostat E fermenter(Braun, Melsungen, Germany) at 57°C. The biomasswas harvested by continuous centrifugation at 17,000g at 4°C (Contifuge 17RS, Heraeus, Germany) andstored at −20°C.

Preparation of cell wall fragmentsThe preparation of cell wall fragments was per-

formed as described in previous studies (12,13). Dur-ing this procedure, whole cells were suspended in 50mM Tris-HCl buffer (pH 7.2) and broken by sonifi-cation with a Branson ultrasonic cell disruptor (Soni-fier 250, Danbury, CT, U.S.A.) at maximum output

for 3 min. Cell wall fragments were separated fromwhole cells by centrifugation at 40,000 g for 15 min at4°C. Plasma membrane fragments were extracted bystirring cell wall fragments in a Triton X-100 solution(0.75% in 50 mM Tris-HCl buffer, pH 7.2) for 20 minat room temperature. After centrifugation, pellets ofcell wall fragments were washed at least 5 times with50 mM Tris-HCl buffer (pH 7.2) and finally withdistilled water.

Preparation of S-layer microparticlesFor crosslinking of the S-layer protein in cell wall

fragments, 1 g wet pellets of cell wall fragments (ob-tained after centrifugation at 40,000 g for 20 min at4°C) were suspended in 100 ml 0.1 M potassiumphosphate buffer (pH 7.2), and 1 ml glutaraldehyde(50% in distilled water) was added. Following a 30min incubation at 20°C, suspensions were centri-fuged at 40,000 g for 10 min at 4°C. The pellets werewashed at least 4 times with distilled water. Subse-quently, Schiff bases were reduced after suspendingpellets in 100 ml sodium borohydride solution (10mM in 10 mM NaOH) at 20°C. After 60 min, sus-pensions were centrifuged again at 40,000 g for 20min at 4°C, and the pellets washed with distilled wa-ter at least 4 times.

Immobilization of Protein ACarboxylic acid groups of the S-layer protein were

activated with water-soluble 1-ethyl-3,3�(dimethyl-amino)carbodiimide (EDC), pH 4.75, at 20°C (15mg/ml distilled water). After 60 min of activation,suspensions were centrifuged at 40,000 g for 15 minat 4°C, and pellets were washed with ice-cold dis-tilled water.

Subsequently, 100 mg wet pellets were suspendedin 2 ml of a Protein A solution (2mg/ml distilledwater, the pH was adjusted to 7.0 with 0.1 N NaOH).The suspensions were stirred at 300 rpm for 4 to 18h at 20°C and then centrifuged at 40,000 g for 20 minat 4°C. In the following, pellets were washed oncewith distilled water and then with 0.1 M phosphatebuffer (pH 7.0) at least 5 times. Protein concentra-tions in the collected supernatants were determinedby the bicinchoninic acid method using Protein A asa standard. Wet weights of pellets were determinedafter centrifugation at 40,000 g for 20 min at 4°C.

Determination of the IgG-binding capacity ofProtein A affinity microparticles

In the following, S-layer microparticles with im-mobilized Protein A are referred to as S-layer basedaffinity microparticles. Wet pellets of S-layer basedaffinity microparticles (100 mg) were suspended in600 �l of FFP and incubated for 60 min at 37°C. FFP

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without S-layer based affinity microparticles wasincubated in parallel as a control. Samples werecentrifuged for 5 min at 18,000 g (Hermle Z233microfuge), and the concentration of IgG in the su-pernatant was determined with a Beckman CoulterArray nephelometer.

In addition, the microparticles from the IgG-binding assay were washed 3 times with 0.9% (w/v)NaCl, and bound IgG was eluted with 4 ml 0.1 Mglycine-HCl buffer (pH 3.5). After centrifugation at18,000 g for 5 min, the amount of IgG in the super-natant was quantified by the Lowry method.

Biocompatibility tests

Sample preparationS-layer microparticles were washed 4 times with

0.9% (w/v) NaCl and suspended in the same solutionin a ratio of 1:10. Following a 20 h incubation at37°C, S-layer microparticles were removed by cen-trifugation for 30 min at 50,000 g and filtrationthrough 0.22 �m filters (MiniSart, Sartorius, Ger-many). The filtrates were tested for limulus amebo-cyte lysate test (LAL)-reactivity, cytotoxicity, andfor the presence of cytokine inducing substances. Acommercially available immunosorbent (IM-PH,Asahi Medical, Tokyo, Japan) was processed in thesame way as the S-layer microparticles and served asa reference in the biocompatibility tests.

LAL-reactivitySamples were assayed for LAL-reactivity using a

kinetic Limulus amebocyte lysate test system(Charles River Endosafe, CoaChrom Diagnostica,Vienna, Austria). Measurements were performed intriplicate.

Toxicity for eukaryotic cellsCytotoxicity of the S-layer microparticles was ex-

amined by a MTT-test (EZ4U, Biomedica, Vienna,Austria). Mouse fibroblasts were incubated with su-pernatants from S-layer microparticles for 90 min at37°C according to the instructions of the manufac-turer. After the addition of the substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT), which is metabolized to coloredproducts only by vital cells, absorbance at 450 nmwas determined. Measurements were performed intriplicate.

Cytokine inducing substancesTwo hundred microliter aliquots of the filtrates of

suspensions containing S-layer microparticles (seeabove) were incubated with 50 �l of fresh humanblood for 20 h at 37°C. Samples were centrifuged for3 min at 5000 rpm, and the tumor necrosis factor �

(TNF�) as well as interleukin 1 receptor antagonist(IL-1 Ra) in the supernatant were quantified byELISA (Biosource, Nivelles, Belgium). Measure-ments were performed in duplicate.

Electron microscopyElectron microscopic investigations were done

with a Philips CM 100 at 80 kV using a 30 �m aper-ture. Negative staining of cell wall fragments andS-layer based affinity microparticles was performedas described (13).

Sodium dodecyl sulfate-polyacrylamidegel electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) was carried out in anXCell II Mini Cell (Novex, Inc., San Diego, CA,U.S.A.) as previously described (14). For the experi-ments, a gel system with a 4% stacking gel and a10% separation gel was used. Gels were stained withCoomassie Blue R-250 or with silver.

RESULTS

Electron microscopic investigation of cellwall fragments

Ultrathin sectioning revealed that cell wall frag-ments from T. thermohydrosulfuricus L111-69 (Fig.1) possess a three-layered envelope profile consist-ing of the outer S-layer, a thin peptidoglycan-con-taining layer, and the inner S-layer. The inner S-layer lattice is formed from a pool of subunits in thepeptidoglycan-containing rigid cell wall layer (12).Characteristic properties of the S-layer from T. ther-mohydrosulfuricus are summarized in Table 1.

Biocompatibility testsFiltrates from S-layer microparticles were tested

for LAL-reactivity, cytotoxicity, and the presence of

FIG. 1. An electron micrograph is shown of negatively stainedaffinity microparticles from T. thermohydrosulfuricus.

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cytokine inducing substances. The samples exhibitedno significant LAL-reactivity (0.017 EU/ml vs. 0.052EU/ml for the reference) and no cytotoxicity (Fig.2). Moreover, incubation of whole blood with fil-trates from S-layer microparticles did not result inelevated cytokine production. The concentrations ofTNF-� and IL-1 Ra in the samples were not higherthan in the blank (whole blood incubated with 0.9%NaCl).

Immobilization capacity of S-layer microparticlesfor Protein A

Protein A was directly bound to EDC-activatedcarboxylic acid groups of the S-layer protein. Bind-ing occurs covalently via peptide bonds. The immo-bilization capacity for Protein A was found to bedependent on the pH of the Protein A solution;maximum values were obtained at pH 7.0 in which29 �g Protein A could be immobilized to 1 mg wetpellet of S-layer microparticles (Fig. 3). This corre-sponds to approximately 9 Protein A molecules perhexameric S-layer unit cell or to a monolayer of Pro-tein A on the surface. It can be expected that ProteinA is immobilized via the amine groups of lysine resi-dues as approximately 50% of the amine groups areaccumulated at one end of the Protein A molecule(15), and it can be assumed that the Protein A mol-

ecules were preferably linked with their long axisperpendicularly to the S-layer lattice (Fig. 4).

Immobilization capacity of S-layer affinitymicroparticles for human IgG

The binding capacity of affinity microparticles forhuman IgG was investigated in batch experiments.The maximum IgG-binding capacity of the particleswas found to lie at 40 mg IgG per g wet pellet (13.4%dry substance). In comparison, a commercially avail-able immunoadsorbent, IM-PH (Asahi Medical, To-kyo, Japan), which binds immunoglobulins mainlyby hydrophobic interactions, adsorbed 7 mg of IgGunder the same experimental conditions. The IgGadsorption capacity of S-layer based affinity micro-particles was also significantly higher than the capac-ity of the commercially available Protein A-silica af-finity matrix Prosorba (Fresenius Hemocare, BadHomburg, Germany) which binds about 8 mg IgG/gmatrix (16). After the batch experiment, bound pro-tein was eluted from the affinity microparticles at pH3.5, and the eluate was subjected to SDS-PAGE.Staining revealed only 1 band at the position of IgGdemonstrating that no significant amounts of otherproteins were removed from human plasma by theaffinity microparticles (Fig. 5).

DISCUSSION

In comparison to conventional affinity particlesthat are composed of a network of polymer chains(and consequently exhibit both, rather broad pore

FIG. 3. Immobilization capacity is shown of S-layer micropar-ticles from T. thermohydrosulfuricus L111-69 for StaphylococcalProtein A at different pH values (40 �g Protein A were appliedper mg wet pellet of S-layer microparticles).

TABLE 1. Characteristic properties of the S-layer from T. thermohydrosulfuricus L111-69 (11)

Number of S-layer subunits per morphological unit 6Surface area occupied by one morphological unit 175 nm2

Surface area of S-layer microparticles based on 0.6 �m diameter and 1.6 �m length 7.2 �m2

Area occupied by the S-layer protein (37.8 �g) present per milligram wet pellet of S-layer microparticles 81 cm2

FIG. 2. Supernatants from S-layer microparticles are not toxic foreukaryotic cells. In the MTT-test applied, the substrate is metabo-lized to a colored product (which is quantified by measurement ofthe absorption at 450 nm) by vital cells only (L111/1 and L111/2:supernatants from S-layer affinity microparticles before and afterincubation, NaCl/1 and NaCl/2: 0.9 % NaCl before and after in-cubation [blank], IM-PH: supernatant from Immunosorba IM-PH[Asahi Medical, Tokyo, Japan] after incubation, GKL929: mousefibroblasts [control]).

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size distribution and random arrangement of func-tional groups), S-layers represent isoporous crystal-line matrices with a pore size of 4 to 5 nm and amolecular exclusion limit of 40,000. Due to the regu-lar arrangement and orientation of functional groupson the S-layer lattice, macromolecules can be immo-bilized in a well-defined and predictable manner. Asmolecules with molecular masses > 40,000 are exclu-sively bound on the outer S-layer surface, immobili-zation is not limited by diffusion. This is in contrastto porous affinity beads in which up to 90 % of theligands are immobilized within the core region (17).

In the present work, S-layer microparticles wereemployed for immobilization of Protein A resultingin an affinity matrix with high adsorption capacityfor human IgG. In biocompatibility tests, filtratesfrom S-layer microparticles exhibited no LAL-reactivity, no cytotoxicity, and contained no signifi-

cant amounts of cytokine inducing substances asshown by a whole blood assay followed by quantifi-cation of TNF� and IL1-Ra.

Currently, our efforts aim at the crystallization ofpurified recombinant S-layer protein on biocompat-ible microparticles, e.g., on cellulose microbeads, tocreate surfaces with high immobilization capacities.This work represents a prerequisite for the combi-nation of S-layer technology and protein engineer-ing. Genetically modified S-layer proteins with in-corporated functional peptide sequences or foreignprotein domains for specific adsorption tasks (e.g.,S-layer protein with binding sites for IgG) could thenbe used for the coating of microparticles to generatespecific adsorbents for various purification purposes.

In summary, adsorbents based on S-layer proteinscan elegantly be combined with the MDS technol-ogy. They offer high surface areas resulting in bothhigh adsorption capacity and high adsorption rates.

Acknowledgments: The excellent technical assistance ofIngrid Linsberger, Eva Rossmanith, and Oliver Stein isgratefully acknowledged. This work was supported by theChristian Doppler Society and in parts by the project P12938-MOB granted by the Austrian Science Foundation.

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