Development of Specific Adsorbents for Human Tumor Necrosis

Biomacromolecules 2005, 6, 1864-1870

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Development of Specific Adsorbents for Human Tumor Necrosis Factor-r: Influence of Antibody Immobilization on Performance and Biocompatibility Viktoria Weber,*,† Ingrid Linsberger,† Marion Ettenauer,† Fritz Loth,‡ Matti Hoÿhtya¨,§ and Dieter Falkenhagen† Christian Doppler Laboratory for Specific Adsorption Technologies in Medicine, Center for Biomedical Technology, Danube University Krems, Krems, Austria, Fraunhofer Institute for Applied Polymer Research, Golm, Germany, and VTT Biotechnology, Espoo, Finland Received October 25, 2004

To develop adsorbents for the specific removal of tumor necrosis factor-R (TNF) in extracorporeal blood purification, cellulose microparticles were functionalized either with a monoclonal anti-TNF antibody (mAb) or with recombinant human antibody fragments (Fab). The TNF binding capacity of the adsorbents was determined with in vitro batch experiments using spiked human plasma (spike: 1200 pg TNF/mL; 1 mg particles in 250 µL plasma). Random immobilization of the full-sized monoclonal antibody to periodateactivated cellulose yielded particles with excellent adsorption capacity (258.1 ( 48.6 pg TNF per mg adsorbent wet weight). No leaching of antibody was detectable, and the adsorbents retained their activity for at least 12 months at 4 °C. We found that the conditions used during immobilization of the antibody (pH, nature of the reducing agent) profoundly influenced the biocompatibility of the resulting adsorbents, especially with respect to activation of the complement system. Particles obtained by random immobilization of the monovalent Fab fragments on periodate-activated cellulose using the same conditions as for immobilization of the mAb exhibited only low adsorption capacity (44 ( 7 pg/mg adsorbent wet weight). Oriented coupling of the Fab fragments on chelate-epoxy cellulose via a C-terminal histidine tag, however, increased the adsorption capacity to 178.3 ( 8.6 pg TNF/mg adsorbent wet weight. Thus, in the case of small, monovalent ligands, the orientation on the carrier is critical to retain full binding activity. Introduction Immobilization of antibodies or antibody fragments on solid surfaces has been studied extensively and is widely applied in modern life sciences, e.g., for affinity chromatography, biosensors, immunoassays, as well as for the development of protein arrays.1-4 Early immobilization techniques which were based on the direct adsorption of antibodies on plastic or glass often resulted in decreased antigen-binding activity and in leaching of the antibody. To overcome these limitations, numerous strategies for covalent immobilization of antibodies have been developed.5-7 Mostly, antibodies are covalently bound to activated supports through exposed amino groups (i.e., lysine side chains) on their surface. As an average antibody molecule contains 60-80 lysine residues, this coupling method results in random antibody orientation. In addition, multipoint attachment of the antibody molecules may lead to decreased activity due to conformational changes or to obstruction of the antigen binding site.8,9 Therefore, site-directed conjugation schemes using cross-linking reagents that specifically react with residues opposite to the antigen binding site have been * To whom correspondence should be addressed. Tel: ++43 2732 893 2632. Fax: ++43 2732 893 4600. E-mail: [email protected]. † Danube University Krems. ‡ Fraunhofer Institute for Applied Polymer Research. § VTT Biotechnology.

developed. However, the oriented immobilization of antibody fragments with various linker systems remains contradictory with reports on both enhanced and reduced antigen-binding activity after the coupling procedure. 10-11 A popular strategy for specifically orienting antibodies on surfaces involves the use of monovalent antibody fragments (Fab) which can either be obtained by enzymatic fragmentation of immunoglobulin G or isolated from antibody libraries by phage display technology.12 Fab contain thiol groups distal to the antigen-binding site which can be utilized for oriented immobilization on appropriately activated support matrixes. Binding is accomplished through the formation of disulfide bridges between the antibody fragments and the activated support. In addition, recombinant antibody technology allows for the preparation of antibody fragments containing tags (e.g., histidine or biotin) at the C-terminus of the molecule, i.e., opposite to the antigen-binding site. Most of the adsorbents that are currently clinically applied for blood purification are selective, i.e., they bind to a range of molecules. Interaction of the target molecules and the adsorption matrix is frequently based on physicochemical properties, such as charge or hydrophobicity. Although it may be desirable for some applications to remove a group of molecules with common characteristics from the plasma (e.g., hydrophobic substances in liver failure, or total IgG in autoimmune diseases with unknown specificity of the

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Adsorbents for Human Tumor Necrosis Factor-R

Figure 1. Schematic drawing of the MDS. The primary circuit containing the patient’s whole blood is separated from the secondary circuit by a plasma filter. In the secondary circuit, plasma is recirculated and kept in suspension together with the adsorbent particles. The purified plasma is filtered back to the primary circuit, and whole blood is reinfused into the patient.

autoantibodies), a drawback of these adsorbents is the unspecific adsorption of plasma proteins or anticoagulants during the treatment. With increasing knowledge of the pathophysiology of certain diseases, there is a trend toward the development of specific adsorbents that target individual factors. A major advantage of this approach is that molecules which do not need to be removed will remain in the plasma, which reduces adverse effects of the therapy. Our work aims at the development of specific adsorbents based on cellulose microspheres which are functionalized with antibodies or antibody fragments. These particles shall be applied in the microspheres-based detoxification system (MDS).13-14 The MDS represents an alternative approach to conventional blood purification systems, in which the patient’s plasma does not perfuse an adsorption column, but is recirculated into the filtrate compartment of the module (Figure 1). Addition of adsorbent microparticles15 to the plasma circuit allows for the rapid removal of pathogenic substances. A major advantage of the system is its high flexibility, as it can be applied for the treatment of various diseases depending on the specificity of the adsorbents used. Furthermore, adsorbents of different specificity can be combined according to the patient’s needs. Potential applications of the MDS include the treatment of autoimmune diseases (adsorption of autoantibodies), rheologic diseases (adsorption of fibrinogen), as well as sepsis (adsorption of inflammatory cytokines, complement factors, and other pathogenic mediators). We wished to develop a generally applicable procedure for the preparation of specific adsorbents and to evaluate the factors contributing to adsorbent efficiency and biocompatibility. Adsorption of tumor necrosis factor-R was chosen as a model, since TNF is a prominent mediator of cellular immune response and inflammation which has been shown to be a key player in the onset of septic shock.16,17 On the other hand, TNF is a pleiotropic factor that may be needed for the resolution of an infection. From this point of view, extracorporeal blood purification has clear benefits over systemic administration of antibodies blocking TNF, as it allows for a modulation of TNF concentration in the circulation. The characteristics of the support matrix used for immobilization of specific ligands profoundly influence the

Biomacromolecules, Vol. 6, No. 4, 2005 1865

performance of the resulting adsorbents. Desirable matrix characteristics are (1) open porous structure with inner surface accessible for target molecules, (2) mechanically stable spherical beads of narrow size distribution, (3) easy derivatization, and (4) chemical stability. In addition, the biocompatibility of adsorbents used for blood purification is critical, as the particles are in direct contact with the patient’s plasma during the treatment. Biocompatibility of an adsorbent is dependent on the characteristics of both support matrix and ligands. Cellulose represents an ideal support matrix, as it combines all of the above-mentioned features. In addition, cellulosic polymers are widely applied in medicine and generally regarded as biocompatible. However, due to its structural similarity to the polysaccharide chains found on bacterial cell walls, cellulose initiates activation of the complement system,18 which evolved as an important element of the innate immune defense against microorganisms and foreign materials. Through generation of the anaphylotoxins C3a and C5a, complement activation leads to a series of secondary inflammatory effects. Therefore, complement compatibility of the adsorbents developed in this study was evaluated by incubation of the particles in whole blood and quantification of C3a release. It is demonstrated that the chemistry used for functionalization of the support matrix profoundly influences the biocompatibility of the resulting particles. Materials and Methods Chemicals. Recombinant human TNF was obtained from R&D Systems (McKinley Place, Minneapolis). Sodium borohydride, sodium meta-periodate, ethylene glycol, epichlorohydrine, and iminodiacetic acid were purchased from Sigma-Aldrich (St. Louis, Missouri). Fresh frozen human plasma was obtained from a local plasma donation center. Cellulose microparticles were provided by the Fraunhofer Institute for Applied Polymer Research (Golm, Germany). Determination of Protein Concentration. Protein concentration was determined with the Biorad Protein Assay (Biorad Laboratories, Hercules, California) using bovine serum albumin as a standard. Preparation of Periodate- Activated Cellulose Beads. Aliquots of 20 g of water swollen cellulose microparticles were mixed with 100 mL of 0.1 M sodium meta-periodate solution. Following gentle shaking of the mixture for 2 h at 40 °C in the dark, ethylene glycol (20 mL) was added and shaking was continued for 1 h. Particles were separated from the supernatant by centrifugation (4000 g) and thoroughly washed with water. The content of dialdehyde groups (corresponding to the degree of activation) was determined by treatment of the particles with sodium hydroxide solution and titration of sodium hydroxide consumption. Typical degrees of activation were in the range of 300-360 µmol per g dry matter. Preparation of Chelate-Epoxy Cellulose Beads. Chelateepoxy cellulose beads were prepared according to a modified method of Pessela et al.19 Aliquots of 10 g of water swollen cellulose microspheres (cellulose content 3 g) were mixed with 25 mL of 2 M sodium hydroxide solution. Epichloro-

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hydrine (12.5 mL) was added and the mixture was shaken for 30 min at 50 °C. Finally, the beads were thoroughly washed with distilled water. The content of epoxy groups (degree of activation) was determined by treatment with sodium thiosulfate and titration of the formed hydroxyl ions with hydrochloric acid. Typical preparations contained about 470 µmol epoxy groups per gram of dry matter. Subsequently, the activated cellulose microspheres were incubated in a solution of 0.2 M iminodiacetic acid (IDA) in 2 M sodium carbonate for 2 h at 25 °C. After washing with distilled water, samples contained approximately 250 µmol epoxy groups per gram of dry matter. Aliquots of 6 g of this sample were resuspended in 40 mL of 50 mM phosphate buffer, pH 6.0, containing 100 mg of CoCl2, incubated for 2 h at 25 °C, and thoroughly washed with distilled water. The content of free epoxy groups was about 170 µmol/g dry matter. The activated microspheres were immediately used for antibody immobilization, as a loss of epoxy groups occurs on storage. Antibodies. A chimeric monoclonal antibody (mAB) against TNF (Remicade, Infliximab) was obtained from the local pharmacy. Infliximab consists of the variable regions of a mouse anti-TNF mAb linked to human IgG1 with κ light chains.20 It binds to the TNF homotrimer with high affinity (Ka ) 1010 M-1) and is currently approved for the treatment of Crohn’s disease and rheumatoid arthritis.21-23 Recombinant human Fab fragments against TNF were produced at VTT Biotechnology using phage display technology. The phage library was constructed from the lymphocytes of 50 volunteer blood donors obtained from the Finnish Red Cross. The library is in single chain format and kappa (2.1 × 107 clones) and lambda (7.4 × 107 clones) light chains are kept in separate libraries both having the same heavy chain variable repertoire. The libraries were selected through five panning rounds against recombinant TNF (Chemicon, Temecula, California) using standard protocols. Four positive clones against TNF were found, three from the kappa library and one from the lambda library. One clone, KC10, was selected for further development. It is composed of VK4/JK1 and VH1/D3/JH3a sequences according to the MRC Centre for Protein Engineering database. The affinity constant of KC10 is 4 × 108 M-1 as determined with BIAcore analysis. The single chain fragments were transformed into Fab fragments by cloning human kappa and human IgG1 CH1 constant regions with overlapping PCR. Two separate affinity tags were introduced, a hexahistidine tag into the C-terminus of the heavy chain and a biotin tag (Avidity, Denver, Colorado) into the C-terminus of the light chain, for oriented coupling of the fragments. The fragments were produced in high-density fed-batch cultures (4-5 liters) in a Bio-Flow IV bioreactor (New Brunswick, Edison, New Jersey) using E.coli host strain RV308 for the histidine and AVB100 for the biotin tagged product. Fragments were purified with Protein G fast flow (Amersham Biosciences, Freiburg, Germany) affinity chromatography. Random Immobilization of Infliximab and Fab on Periodate-Activated Cellulose Beads. Periodate-activated cellulose particles (30 mg wet weight) were washed 3 times

Weber et al. Table 1. TNF Adsorption Capacities (pg TNF/mg Adsorbent Wet Weight) of Microparticles Obtained by Random Immobilization of the Full-Sized Monoclonal Antibody on Periodate-Activated Cellulose at Different Values of pH (Mean of 3 Experiments ( SD) pH

TNF adsorption (pg/mg adsorbent wet weight) ( SD

9.5 7.4 5.5

258.1 ( 49 210 ( 32 98 ( 26

Table 2. Coupling Efficiencies and Amounts of TNF Bound for Immobilization of Fab to Periodate-Activated Cellulose (PAC) and Chelate-Epoxy Cellulose (CEC)a amount of Fab used TNF bound to for coupling to coupling Fab immobilized immobilized 30 mg beads efficiencyb on the particles Fab matrix (µg) (%) (µg per mg) (pg per µg) PAC PAC PAC CEC CEC CEC

500 1000 2500 500 1000 2500

100 57 23 100 80 35

17.5 ( 0.9 20 ( 2.4 21 ( 3.1 17.5 ( 0.7 28 ( 2.2 29 ( 1.8

1.7 ( 0.1 1.6 ( 0.2 1.9 ( 0.2 5.9 ( 0.6 5.7 ( 0.4 6.1 ( 0.5

a Data are given as means of 3 experiments ( SEM. b Calculated from the amount of protein (i.e., antibody) in the supernatant before and after coupling.

with phosphate buffered saline (PBS; pH 7.4). Particles were separated from the supernatant by centrifugation (3 min, 4000 g). After a final wash with 0.1 M sodium borate (pH 9.5), the particles were suspended in 6 mL of 0.1 M sodium borate (pH 9.5), and 100 µL of a solution containing 10 mg/mL Infliximab in PBS was added. The mixture was briefly vortexed and incubated overnight (16-20 h) at 37 °C with gentle shaking. Beads were separated from the supernatant by centrifugation and washed with PBS. After complete removal of the supernatant, 100 µL of sodium borohydride or sodium cyanoborohydride, respectively, (2 mg/mL in PBS) was added to reduce Schiff bases. After incubation for 60 min at room temperature, particles were removed by centrifugation and washed 3 times with PBS. The adsorbents were suspended in PBS and kept at 4 °C until further analysis. Coupling efficiency was calculated from the amount of protein (i.e., antibody) in the supernatant before and after coupling. Typically, between 30 and 35% of the full-sized antibody used in an experiment were immobilized on the beads. Fab fragments were randomly immobilized on periodate-activated cellulose exactly as described for Infliximab using 500, 1000, and 2500 µg of Fab per 30 mg (wet weight) of activated cellulose. Coupling efficiencies for Fab are given in Table 2. Leaching of Full-Sized Monoclonal Antibody. Adsorbent suspensions were centrifuged (4000 g, 3 min) and supernatants were assayed for leached antibody by human anti-κ light chain ELISA (Bethyl Laboratories, Montgomery, Texas). Oriented Immobilization of Histidine-Tagged Fab on Co-Chelate Epoxy Cellulose Beads. Immobilization of Fab on Co-chelate epoxy beads was performed according to Pessela et al.19 (Scheme 1). Aliquots of the particles (30 mg

Adsorbents for Human Tumor Necrosis Factor-R


Scheme 1: Oriented Immobilization of Histidine-Tagged Fab Fragments on Chelate-Epoxy Cellulosea

a Cellulose microparticles are activated with epichlorohydrine and incubated with iminodiacetic acid to introduce IDA into the support. IDA-epoxy cellulose is then incubated in the presence of cobalt salts to obtain chelate-epoxy cellulose. This support is used to immobilize the histidine-tagged Fab fragments in an oriented manner.

wet weight) were incubated overnight with 500, 1000, and 2500 µg of histidine-tagged Fab in 30 mM phosphate buffer (pH 7.0). Beads were separated from the supernatant by centrifugation (4000 g, 3min) and treated with 5 mL of 1M glycine (pH 8.5) for 4 h at room temperature to block residual epoxy groups. Adsorbents were washed and stored in 30 mM phosphate buffer (pH 7.0) at 4 °C. Adsorption of TNF. Binding of TNF to the different adsorbents was evaluated in batch-experiments using spiked human plasma (1200 pg TNF /ml). Adsorbents were mixed with freshly spiked human plasma (1 mg of particles in 250 µL of plasma) and incubated in an end-over-end shaker for 60 min. Samples from the supernatant were taken after 5, 30, and 60 min of incubation, and TNF was quantified by ELISA (Biosource, Nivelles, Belgium). Activation of the Complement System. Blood was drawn from healthy volunteers and anticoagulated with heparin (3 IU/mL). Adsorbent particles were incubated in freshly drawn blood at 37 °C using 100 mg of adsorbent and 1900 µL of blood. Samples were taken after 5 and 60 min of incubation, respectively, and the reaction was stopped by addition of 80 µL of ice-cold sodium-EDTA (30% w/v in 0.9% NaCl). Particles were removed by centrifugation, and C3adesArg as a marker of complement activation was quantified in the supernatant by ELISA (Progen, Heidelberg, Germany). Results and Discussion Cellulose Microparticles as Starting Material for Activation. Figure 2 shows an electron micrograph of the cellulose particles used as starting material both for oriented

Figure 2. Electron micrograph of cellulose microparticles used as carriers for antibodies and antibody fragments, respectively.

and random immobilization of the antibodies. The particles are highly porous and in a size range of 1-10 µm. Particle size distribution remained unchanged upon recirculation of a suspension (50% v/v of particles in 0.9% saline) at a flow rate of 4L/min for 30 h, proving that the particles are mechanically stable. Immobilization of Full-Sized Antibody on Periodate Activated Cellulose. Adsorption capacities of particles obtained by random immobilization of the full-sized monoclonal antibody at different values of pH are summarized in Table 1. Adsorption capacity was highest for particles obtained by coupling at pH 9.5 according to the protocol described in Materials and Methods. However, high pH during the coupling reaction led to particles which strongly

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activated the complement system (see below). Therefore, further immobilization reactions were performed at pH 7.4. To assess their stability, adsorbent suspensions were stored in PBS (pH 7.4, 0.3% sodium azide), and samples were analyzed for leaching of antibody and TNF binding capacity after 3, 6, and 12 months of storage, respectively. No loss of activity was observed over a period of at least 12 months. Leaching of antibody from the particles was tested by analyzing supernatants of particle suspensions for human κ-light chains. Antibody concentration in the supernatant remained below the detection limit of 40 ng/mL in all samples. Stability of Chelate-Cellulose Microparticles. To assess the stability of the chelate-cellulose microparticles, samples from consecutive activation steps (before and after reaction with IDA and after addition of Co2+) were stored at 4 °C and at 23 °C, respectively, and their epoxy group content was analyzed over time. Starting from 470 µmol/g, there was a slow but constant decrease in epoxy group content which reached a plateau at 390 and 310 µmol/g at 4° and 23 °C, respectively, after 300 h of storage. The starting level of epoxy groups in the IDA treated sample was 250µmol/g, as about half of the epoxy groups had already been consumed for binding of IDA. Again, the epoxy group content decreased over time to 200 µmol/g after 300 h at 4 °C. Addition of Co2+ to the IDA treated sample resulted in a further significant decrease in epoxy groups from 250 to 150 µmol/g indicating that Co2+ might exert a catalytic effect on the hydrolysis of epoxy groups. Therefore, immobilization of protein ligands should be carried out immediately after activation of the cellulose particles to avoid excessive loss of epoxy groups. After immobilization of the Fab fragments, the resulting adsorbents fully retained their activity for at least 6 months. Adsorption of TNF: Random versus Oriented Immobilization of Full-Sized Antibody and Fab Fragments. Random immobilization of the full-sized antibody to periodate-activated cellulose beads yielded particles with high TNF adsorption capacity (258 ( 49 pg TNF/mg adsorbent wet weight) as well as high adsorption rate (Figure 3). In contrast, particles obtained by random immobilization of Fab to periodate-activated cellulose exhibited only weak TNF adsorption. Immobilization of 500, 1000, and 2500 µg of Fab per 30 mg of particles resulted in coupling efficiencies of 100, 57, and 23%, respectively, corresponding to 17.5, 20, and 21 µg of Fab immobilized per mg of particles. Thus, use of higher amounts of Fab (2500 µg vs 500 µg) did not result in significantly increased immobilization of Fab (Table 2). TNF adsorption was 44 ( 7 pg/mg adsorbent wet weight after 60 min for the series using 2500 µg Fab/30 mg particles (Figure 3). For Fab immobilized to chelate-epoxy cellulose using 500, 1000, and 2500 µg of Fab per 30 mg of particles, coupling efficiencies were 100, 80, and 35%, respectively, corresponding to 17.5, 28, and 29 µg of Fab immobilized per mg of particles (Table 2). Oriented coupling considerably improved TNF binding capacity to 178.3 ( 8.6 pg TNF/mg adsorbent wet weight. In comparison, 6.1 pg of TNF were adsorbed per µg of Fab immobilized on chelate-epoxy

Weber et al.

Figure 3. Adsorption of TNF by specific adsorbents in batch experiments. Samples were taken after 5, 30, and 60 min of incubation and TNF was quantified by ELISA. Ctrl, unmodified cellulose; Mab-random, monoclonal antibody randomly immobilized to periodate-activated cellulose (PAC); Fab-random, Fab fragments randomly immobilized to PAC; Fab-oriented, Fab in oriented immobilization on chelate-epoxy cellulose. Both for Fab-random and Fab-oriented, the results for particles obtained by coupling 2500 µg of Fab per 30 mg of cellulose are shown. Data are given as mean of three experiments ( SEM.

Figure 4. Influence of coupling conditions on complement activating activity of the adsorbents. Generation of C3adesArg was quantified after incubation of blood with (1) untreated cellulose microparticles, (2) PAC; (3) PAC after immobilization of monoclonal antibody at pH 7.4 and reduction with sodium cyanoborohydride; (4) PAC after treatment with sodium cyanoborohydride, but without antibody (pH 7.4); (5) PAC after immobilization of antibody and reduction with sodium cyanoborohydride at pH 9.5; (6), as (5), but with borohydride instead of cyanoborohydride.

cellulose, as compared to 1.9 pg TNF per µg of Fab immobilized on periodate-activated cellulose. As shown in a control experiment, unmodified cellulose microparticles exhibited only negligible binding of TNF. Influence of Coupling Conditions to Periodate-Activated Cellulose on Biocompatibility. To measure the complement activating activity of the cellulose-based TNF adsorbents, generation of C3adesArg was quantified after incubation of the adsorbents in whole blood. As shown in Figure 4, complement activation was in the same range for periodate-activated cellulose as for untreated cellulose. Two parameters during the antibody immobilization procedure were found to significantly influence the complement activating activity of the adsorbents, namely (1) the nature

Adsorbents for Human Tumor Necrosis Factor-R Scheme 2: Reduction of Periodate-Activated Cellulose with Borohydride and Cyanoborohydride, Respectivelya

a Borohydride is specific for the Schiff base structure and does not affect the original aldehyde groups. By contrast, cyanoborohydride converts unreacted aldehydes into hydroxyls in addition to Schiff base reduction.

of the reductant used for stabilization of Schiff bases (sodium borohydride vs sodium cyanoborohydride) and (2) the pH during the coupling reaction. Adsorbents obtained by reduction with borohydride exhibited significantly higher complement activation than particles treated with cyanoborohydride. Borohydride reduces aldehyde groups to hydroxyls and converts Schiff bases to secondary amines, whereas cyanoborohydride is milder24 and effects Schiff base reduction without reducing aldehydes (Scheme 2). We hypothesize that it is the generation of additional neighboring hydroxyl groups during the reduction with borohydride that triggers complement activation. This is supported by the finding that deposition of the complement protein C3b on biomaterials increases with increasing surface hydroxyl density.25 Regarding the influence of the pH during the coupling reaction, complement activation was strongly enhanced in adsorbents produced at pH 9.5 as compared to pH 7.4. This may be explained by the fact that, due to cleavage of the C2-C3bonds of the glucose rings, the cellulose backbone is weakened after activation with periodate and therefore becomes susceptible to high pH. For this reason, further coupling reactions were performed at pH 7.4. Remarkably, complement activation was attenuated after immobilization of the antibody (Figure 5, bars 3 vs 4). Conclusions Covalent immobilization of a full-sized anti-TNF antibody or of Fab fragments specific for TNF to cellulose microparticles yielded adsorbents that were highly efficient both in terms of adsorption capacity and kinetics. The full-sized antibody exhibited excellent binding capacity after random immobilization to periodate activated cellulose, indicating that the orientation of the ligand on the support was not critical for performance of the adsorbent. In contrast, Fab fragments immobilized under identical conditions resulted in very low adsorption capacity. Although this may partly be attributed to their lower affinity constant for TNF as compared to the full-sized antibody, we demonstrated that


oriented immobilization of the antibody fragments led to a significant increase in binding capacity of the resulting adsorbents implying that, for small, monovalent antibody fragments, the orientation on the carrier is critical. Recombinant antibody technology offers various possibilities to introduce tags for oriented immobilization into the antibody fragments. In our study, a C-terminal histidine tag was used in combination with iminodiacetic acid-epoxy activation of the cellulose microparticles. In a two-step reaction, the histidine tag interacts with immobilized iminodiacetic acid leading to correct orientation of the Fab on the surface prior to formation of covalent bonds via amino groups of the antibody. For random immobilization on periodate activated cellulose, it was shown that the biocompatibility of the adsorbents depends on the characteristics of both support matrix and ligand, as well as on the conditions used during the immobilization reaction. In summary, the attachment of antibodies or antibody fragments to activated cellulose microparticles offers a method to produce highly efficient and specific adsorbents for the removal of a variety of pathogenic factors in extracorporeal blood purification. Acknowledgment. This work was supported by the Christian Doppler Forschungsgesellschaft. References and Notes (1) Peluso P.; Wilson, D. S.; Do, D.; Traan, H.; Venkatasubbaiah, M.; Quincy, D.; Heidecker, B.; Poindexter, K.; Tolani, N.; Phelan, M.; Witte, K.; Jung, L. S.; Wagner, P.; Nock, S. Optimizing antibody immobilization strategies for the construction of protein microarrays. Anal. Biochem. 2003, 312, 113-124. (2) Vankova´, R.; Gaudinova´, A.; Su¨ssenbekova´, H.; Dobrev, P.; Strnad, M.; Holı´k, J.; Lenfeld, J. Comparison of oriented and random antibody immobilization in immunoaffinity chromatography of cytokinins. J. Chromatogr. A 1998, 811, 77-84. (3) Lu, B.; Smyth M. R.; O’Kennedy, R. Oriented immobilization of antibodies and its applications in immunoassays and immunosensors. Analyst 1996, 121, 29R-32R. (4) Reimer, U.; Reineke, U.; Schneider-Mergener, J. Peptide arrays: from macro to micro. Curr. Opin. Biotechnol. 2002, 13, 315-320. (5) Danczyk, R.; Krieder, B.; North, A.; Webster, T.; HogenEsch, H.; Rundell, A. Comparison of antibody functionality using different immobilization methods. Biotechnol. Bioeng. 2003, 84 (2), 215223. (6) Brogan, K. L.; Wolfe, K. N.; Jones, P. A.; Schoenfisch, M. direct oriented immobilization of F(ab’) antibody fragments on gold. Anal. Chim. Acta, 2003, 496, 73-80. (7) Wimalasena, R. L.; Wilson, G. S. Factors affecting the specific activity of immobilized antibodies and their biologically active fragments. J. Chromatogr. 1991, 572, 85-102. (8) Subramanian, A.; Velander, W. H. Effect of antibody orientation on immunosorbent performance. J. Mol. Recognit. 1996, 9, 528-535. (9) Turkova´, J. Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function. J. Chromatogr. B 1999, 722, 11-31. (10) Wilchek, M.; Miropn, T. Oriented versus random protein immobilization. J. Biochem. Biophys. Methods 2003, 55, 67-70. (11) Reference deleted in revision. (12) Hoogenboom, H. R. Overview of antibody phage-display technology and its applications. Methods Mol. Biol. 2002, 178, 1-37. (13) Falkenhagen, D.; Schima, H.; Loth, F. Arrangement for removing substances from liquids, in particular blood. United States Patent, # 5,855,782, 1999. (14) von Appen, K.; Weber, C.; Losert, U.; Schima, H.; Gurland, H. J.; Falkenhagen, D. Microspheres based detoxification system: a new method in convective blood purification. Artif. Organs. 1996, 20, 420-425.

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(15) Vo¨llenkle, C.; Weigert, S.; Ilk, N.; Egelseer, E.; Weber, V.; Loth, F.; Falkenhagen, D.; Sleytr, U. B.; Sa´ra, M. Construction of a functional S-layer fusion protein comprising an immunoglobulin G-binding domain for development of specific adsorbents for extracorporeal blood purification. Appl. EnViron. Microbiol. 2004, 1514-1521. (16) Fiers, W. Tumor necrosis factor. Characterization at the molecular, cellular, and in vivo level. FEBS Lett. 1991, 285, 199-212. (17) Tracey, K. J.; Cerami, A. Tumor necrosis factor: a pleiotropic cytokine and therapeutic target. Annu. ReV. Med. 1994, 45, 491-503. (18) Johnson R. J. Complement activation during extracorporeal therapy: biochemistry, cell biology and clinical relevance. Nephrol. Dial. Transplant. 1994, 9, 36-45. (19) Pessela, B. C. C.; Mateo, C.; Carrascosa, A. V.; Vian, A.; Garcıá J. L.; Rivas, G.; Alfonso, C.; Guisan, J. M.; Fernańdez-Lafuente, R. One-Step Purification, Covalent Immobilization, and Additional Stabilization of a Thermophilic Poly-his-Tagged β-Galactosidase from Thermus sp. Strain T2 by using Novel Heterofunctional ChelateEpoxy Sepabeads. Biomacromolecules 2003, 4, 107-113. (20) Knight, D. M.; Trinh, H.; Le, J.; Siegel, S.; Shealy, D.; McConough, M.; Scallon, B.; Moore, M. A.; Vilcek, J.; Daddona, P.; Ghrayeb, J.

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Construction and initial characterization of a mouse-human chimeric anti-TNFantibody. Mol. Immunol. 1993, 30, 1443-1453. Targan, S. R.; Hanauer, S. B.; van Deventer, S. J.; Mayer, L.; Present, D. H.; Braakman, T.; DeWoody, K. L.; Schaible, T. F.; Rutgeerts, P. J. A short-term study of the chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn’s disease. N. Engl. J. Med. 1997, 337 (15), 1029-1035. Klinkhoff, A. Biological agents for rheumatoid arthritis: targeting both physical function and structural damage. Drugs 2004, 64, 126783. Olsen, N. J.; Stein, C. M. New drugs for rheumatoid arthritis. N. Engl. J. Med. 2004, 350 (21), 2167-79. Peng, L.; Calton, G. J.; Burnett, J. W. Effect of borohydride reduction on antibodies. Appl. Biochem. Biotechnol. 1987, 14, 91-99. Hirata, I.; Hioki, J.; Toda, M.; Kitazawa, T.; Murakami, Y.; Kitano, E.; Kitamura, H.; Ikada, Y.; Iwata, H. Deposition of complement protein C3b on mixed self-assembled monolayers carrying surface hydroxyl and methyl groups studied by surface plasmon resonance. J. Biomed. Mater. Res. 2003, 66A, 669-676.

BM040074T

Development of Specific Adsorbents for Human Tumor Necrosis

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