Development of specific adsorbents for human tumor necrosis factor-alpha: influence of antibody immobilization on performance and biocompatibility.
To develop adsorbents for the specific removal of tumor necrosis factor-alpha (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 muL plasma). Random immobilization of the full-sized monoclonal antibody to periodate-activated 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 degrees 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.
Weber
Viktoria
V
Christian Doppler Laboratory for Specific Adsorption Technologies in Medicine, Center for Biomedical Technology, Danube University Krems, Krems, Austria. viktoria.weber@donau-uni.ac.at
Linsberger
Ingrid
I
Ettenauer
Marion
M
Loth
Fritz
F
Höyhtyä
Matti
M
Falkenhagen
Dieter
D
D000327:Adsorption; D000906:Antibodies / Q000276:immunology*; D001672:Biocompatible Materials; D002482:Cellulose / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008854:Microscopy, Electron; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000276:immunology
D000906:Antibodies; D001672:Biocompatible Materials; D007140:Immunoglobulin Fab Fragments; D014409:Tumor Necrosis Factor-alpha; D002482:Cellulose