Effects of Synchronization on CD40 Expression and Antibody Production in Hybridoma Cells Stimulated with Anti-mIgG

Abstract

In previous experiments with the 55-6 hybridoma cell line, we showed that cell stimulation with anti-mouse surface immunoglobulin G antibody (anti-mIgG) increased both CD40 expression and specific monoclonal antibody (mAb) production rate. Cell preincubation with lipopolysaccharide (LPS) prior to anti-mIgG stimulation enhanced these results. Moreover, the expression of both CD40 and surface immunoglobulin G (sIgG) were higher for cells in the G1 phase of the cell cycle. Therefore, to determine the relationship between cell cycle position, CD40 expression, and mAb productivity, in this work cells were synchronized in the G1 phase by thymidine block. In addition, synchronized cells were subjected to different treatments with anti-mIgG. Although synchronized cells showed a slight increase in both CD40 expression and maximum specific growth rate (mu max) compared with unsynchronized cells, specific productivity did not show significant changes. However, the stimulation of synchronized cells with anti-mIgG increased over 65% the expression of CD40 and over 50% the specific productivity in comparison with that obtained on unsynchronized cells after anti-mIgG stimulation. These data improved additionally over 15 and 60%, respectively, by adding 2 mM thymidine to the culture medium. These results suggest that the effect of the positive association between G1 phase, CD40 expression, and specific productivity is subordinated to the effect of anti-mIgG stimulation, which is enhanced by increasing the percentage of cells on the G1 phase of the cell cycle. Contrary to expectations, LPS preincubation of synchronized cells prior to anti-mIgG stimulation did not increase the specific productivity in comparison with non-preincubated cells, and the expression of CD40 was minor compared to that on non-preincubated cells.

Model‐based identification of cell‐cycle‐dependent metabolism and putative autocrine effects in antibody producing CHO cell culture

“…An increase of the antibody production has been mostly described during the G0 or G1 phase for hybridoma or CHO cells (Becerra, Berrios, Osses, & Altamirano, ; Martin‐Lopez, Garcia‐Camacho, Contreras‐Gomez, & Molina‐Grima, ; Park, Noh, Woo, Kim, & Lee, ; Suzuki & Ollis, ). In contrast to our study, temperature shifts or butyrate treatment were used to arrest the cell cycle in the G1 phase and therefore enrich G0 cells with assumed higher productivity in the stationary phase, in which most of the product is formed (Du et al, ; Hendrick et al, ; Münzer et al, ; Wippermann et al, ).…”

Enhanced Monoclonal Antibody Production in Hybridoma Cells by LPS and Anti‐mIgG

“…Effect of LPS in Combination with Anti‐Mouse IgG on CD69, CD40, and CD19 Expression. In a previous study (18) we examined activation of the 55‐6 cell line with anti‐mIgG. It was demonstrated that the effect was anti‐mIgG dose‐dependent, and the best results were found at a concentration of 3,700 pg cell −1 .…”

“…It was found that concentration of free anti‐mIgG initially added to the culture medium decreased progressively over time due to the capture of anti‐mIgG by secreted IgG2a (18) and at about 60 h was exhausted (Figure 4). The decrease in CD69, CD40, and CD19 expression over time was in agreement with the decrease in free anti‐mIgG concentration.…”

“…The weak effect of the anti‐mIgG was misleading, since the free anti‐mIgG initially added to the culture medium was bound to IgG2a, as this was secreted by the cells (18), and its concentration decreased progressively until its total exhaustion after 60 h. Figures 4 and 7 show how the marked decrease in CD69, CD40, and CD19 expression (Figure 4) and in specific IgG2a production rate (Figure 7B) coincide with exhaustion of free anti‐mIgG. Strategies focused on maintaining high levels of anti‐IgG in the culture medium would lead to larger increases in the specific IgG2a production rate.…”

“…IgG2a concentration was measured by the sandwich‐type ELISA using goat anti‐mouse IgG‐coated plates and goat anti‐mouse IgG peroxidase conjugate as the second antibody. The capture of secreted IgG2a by free anti‐mIgG antibody added in culture medium was determined as described elsewhere (18).…”