Selective improvement of tumor necrosis factor capture in a cytokine hemoadsorption device using immobilized anti-tumor necrosis factor

Abstract

Sepsis is a harmful hyper-inflammatory state characterized by overproduction of cytokines. Removal of these cytokines using an extracorporeal device is a potential therapy for sepsis. We are developing a cytokine adsorption device (CAD) filled with porous polymer beads which efficiently depletes middle-molecular weight cytokines from a circulating solution. However, removal of one of our targeted cytokines, tumor necrosis factor (TNF), has been significantly lower than other smaller cytokines. We addressed this issue by incorporating anti-TNF antibodies on the outer surface of the beads. We demonstrated that covalent immobilization of anti-TNF increases overall TNF capture from 55% (using unmodified beads) to 69%. Passive adsorption increases TNF capture to over 99%. Beads containing adsorbed anti-TNF showed no significant loss in their ability to remove smaller cytokines, as tested using interleukin-6 (IL-6) and interleukin-10 (IL-10). We also detail a novel method for quantifying surface-bound ligand on a solid substrate. This assay enabled us to rapidly test several methods of antibody immobilization and their appropriate controls using dramatically fewer resources. These new adsorbed anti-TNF beads provide an additional level of control over a device which previously was restricted to nonspecific cytokine adsorption. This combined approach will continue to be optimized as more information becomes available about which cytokines play the most important role in sepsis.

FIGURE 1

Bead modification chemistry: oxidation of the PSDVB portion of CytoSorb^™ beads to incorporate carboxyl groups. Note that this schematic does not account for the presence of the PVP coating.

FIGURE 2

EDC activation chemistry: preparing carboxyl groups for covalent attachment to exposed amine groups on antibodies.

FIGURE 3

Experimental setup of cytokine capture experiments. The CAD is connected in line with a peristaltic pump and a reservoir of serum spiked with one or more cytokines.

FIGURE 4

Anti-IgG-HRP surface density on beads containing adsorbed antibodies, beads containing covalently bound antibodies, and beads containing PLL-crosslinked antibodies. (n = 3 for each type of bead).

FIGURE 5

TNF removal during 4 h recirculation using CADs packed with unmodified beads containing no antibodies (◆), beads containing covalently bound anti-TNF (○), and beads containing adsorbed anti-TNF (□). (n = 3 for each type of beads).

FIGURE 6

TNF removal during 4-h recirculation using CADs packed with unmodified beads containing no antibody (◆), beads containing adsorbed anti-TNF (□), and beads containing adsorbed IgG (○). (n = 3 for each type of beads).

FIGURE 7

Comparison of the simultaneous removal of IL-6 (a), IL-10 (b), and TNF (c) using unmodified beads containing no antibodies (◆) and beads containing adsorbed anti-TNF antibodies (□). (n = 3 for both types of beads).

FIGURE 8

Rate of anti-IgG-HRP leaching (◆) and overall amount lost (□) during 2-h PBS/BSA flush of beads containing adsorbed antibody. This flushing step was performed prior to the cytokine capture shown in Figure 9. (n = 3).

FIGURE 9

Comparison of TNF removal using beads containing adsorbed anti-TNF with (◆, solid line) and without (□, dashed line) 2-h preflush. The results of the flushing step performed on the beads used in these experiments can be seen in Figure 8. (n = 3 for both types of beads).