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.
The aim of this work was to synthesize and characterize cellulose microspheres with a particle size below 5 lm and narrow size distribution. After activation and functionalization with antibodies, these particles shall be applied as adsorbents in suspensionbased extracorporeal blood purification systems, such as the Microspheres-Based Detoxification System. In the frame of this work such microspheres were developed and synthesized with reproducible properties. Besides using well-established methods for characterization of this kind of bead cellulose, additional procedures for the examination of its properties were developed and applied.
In the frame of this work, cellulose microbeads with an average particle size of 2.3 lm were characterized with respect to porosity using a batch solute exclusion method and two groups of model substances, namely proteins and polystyrene sulfonates. The pores of the microbeads were almost completely accessible to proteins with Stokes radii below 2.5 nm. More than 60% of the pores were accessible to albumin, which is relevant for the application in blood purification, since many target substances are albumin bound. Activation of the microbeads with increasing amounts of sodium metaperiodate yielded matrices with dialdehyde contents between 100 and 1,000 lmol/g. The activated beads were well suited for the covalent attachment of functional ligands, such as antibodies. Immobilization of antibodies against the pro-inflammatory cytokine TNF-a resulted in efficient TNF-a adsorbents which possess application potential in extracorporeal blood purification, e.g. for the modulation of cytokine levels as supportive therapy for sepsis and other inflammatory disorders.
The microspheres-based detoxification system (MDS) is a combined membrane-adsorption system for extracorporeal blood purification in which adsorbent microparticles are recirculated in an extracorporeal filtrate circuit. Because the plasma filter represents the only barrier between the adsorbents and the patient's blood, there is the potential risk of particle entrance into the patient in case of a membrane rupture. To guarantee first fault safety of the system required for clinical application, magnetic fluorescent microparticles are added as markers to the adsorbent circuit. Detection of these particles in the venous blood line results in immediate shutdown of the pumps. Magnetic beads were functionalized with cresyl violet and tested with an in vitro setup of the particle detector to assess the detection limit in different matrices (water versus blood) as well as the influence of flow rate and particle size on the signal. In addition, biocompatibility and influence of sterilization on the performance of the particles were assessed. Functionalization of the magnetic particles with cresyl violet yielded fluorescent particles that were stable at 4 degrees C for at least 12 months. No leakage of dye was detectable, and the particles were neither cytotoxic nor mutagenic. The particles could be steam sterilized without significant loss in fluorescence intensity. With an in vitro setup of the particle detector, 0.1 mg and 5 mg of particles were reproducibly detectable in water and blood, respectively.
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