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We studied the role of CD28 in T-cell biology and T cell-mediated pathology using a novel mouse anti-mouse CD28 antibody, E18, which recognizes an epitope close to the B7 binding site. In vitro, this antibody completely blocked binding of B7 molecules to CD28 expressed on mouse thymocytes but enhanced anti-CD3-induced proliferation of peripheral T cells. Injections of E18 monoclonal antibody into normal BALB/c mice in vivo, however, led to a reversible reduction in Treg cell frequencies among CD4(+) cells, both in the thymus and in secondary lymphoid organs, suggesting that E18 acted as an inhibitor of CD28 signaling under these conditions. Antagonistic activity of E18 in vivo was further implied by suppressed responses of conventional CD4(+) T cells to stimulation with the superantigen staphylococcal enterotoxin B and in a model of acute graft-versus-host disease. In contrast to healthy mice, intact monoclonal antibody E18, but not its nonstimulatory Fab fragment, increased the frequencies of Treg cells among CD4(+) T cells in these pro-inflammatory settings allowing for efficacious protection from acute graft-versus-host disease. Thus, the agonistic signal generated by conventional, ie, nonsuperagonistic, anti-CD28 antibodies is important for their immunotherapeutic potential in vivo.

Expanded bed adsorption (EBA) was examined as the initial capture/purification step in the purification of monoclonal antibodies from Chinese hamster ovary (CHO) cultures. Two process alternatives each using EBA were compared to a conventional Protein A process without EBA. One alternative used Protein A affinity EBA followed by packed-bed cation and anion-exchange steps. The other alternative used cation-exchange EBA as the capture step followed by packed-bed Protein A and anion-exchange steps. The process using Protein A EBA produced comparable purity (host cell protein, DNA, Protein A, antibody aggregate) to the conventional process. However, the Protein A EBA column showed a significant decrease in dynamic capacity with a limited number of cycles. The process using cation EBA achieved comparable levels of host cell proteins (HCP) and DNA but not antibody aggregate or leached Protein A compared to the conventional process.

We show that expanded bed protein A affinity chromatography using Streamline rProtein A media is an efficient method for purifying a recombinant humanized monoclonal antibody from unclarified Chinese hamster ovary cell culture fluid and that it provides purification performance comparable to using a packed bed. We determined that the dynamic capacity of the expanded bed media is related to flow rate (measured in column volumes per hour) by a power function, which allows a high capacity at a low flow rate. At 250 cm h-1 with a 25 cm bed height (10 column volumes h-1), the dynamic capacity is 30 g l-1. The yield and purity (measured by the amount of host cell proteins, DNA, SDS-PAGE, and turbidity) of the antibody purified by expanded bed is comparable to the yield and purity obtained on a standard packed bed method using Prosep A media.

We describe the performance characteristics of five Protein A affinity-chromatography sorbents (Sepharose Fast Flow, Poros 50, Poros LP, Prosep and Streamline) for purifying a recombinant humanized monoclonal antibody from clarified Chinese hamster ovary cell culture fluid. We measured the dynamic capacity at varying flow rates, maximum capacity, pressure drop and production rate. For purified antibody, we measured yield and purity (by SDS/PAGE, the amount of DNA, the amount of host-cell proteins and the amount of Protein A). We found that, whereas all sorbents provided significant and essentially equivalent antibody purification, there were differences in capacity and pressure drop, which affected the production rate and had implications for process applications.

Purpose: Cardiopulmonary bypass is associated with activation of neutrophils, which may adhere to vascular endothelium causing lung, heart, and brain injury. We tested whether blocking neutrophil adherence would improve organ function following cardiopulmonary bypass in dogs., Materials and Methods: All dogs received a standard anesthetic, and then one group (n = 6) received 2 hours of cardiopulmonary bypass followed by 4 hours of observation. A second group (n = 6) received a monoclonal antibody (6 mg/kg) to CD18, a neutrophil adherence factor, immediately before cardiopulmonary bypass. A third group (n = 6) did not receive cardiopulmonary bypass or antibody., Results: Using flow cytometry we found that the antibody bound essentially all neutrophil CD18 sites. All three groups had similar gas exchange and hemodynamics. Lung and heart histology results were similar between groups. By echocardiography, five animals receiving cardiopulmonary bypass alone showed regional wall abnormalities, whereas only one receiving antibody showed wall motion abnormality (P < .05). Following cardiopulmonary bypass, intracellular myocardial pH was higher (P < .05) in the antibody-treated group compared with the group that had cardiopulmonary bypass alone (7.23 +/- 0.05 v 7.07 +/- 0.07 respectively)., Conclusion: Monoclonal antibodies to CD18 can prevent the deterioration in cardiac function routinely observed following cardiopulmonary bypass.

High-performance liquid affinity chromatography (HPLAC) utilizing Protein G as a ligand has been evaluated for rapid quantification of monoclonal antibodies (MAbs) in various solutions. The results obtained by HPLAC agreed to within 10% of a standard enzyme-linked immunospecific assay (ELISA). A standard curve was prepared by injection of known amounts of a purified murine IgG1 with the elution peak area analyzed by computer integration software. Accuracy of quantification was independent of the injection volume, solution compositions, or mouse IgG subclass. A method is described for using Protein G HPLAC to determine murine IgG levels in various complex mixtures within 15 min, compared to the ELISA which required 5 h.