Inhibitory effects of persistent apoptotic cells on monoclonal antibody production in vitro: simple removal of non-viable cells improves antibody productivity by hybridoma cells in culture

Abstract

Cells undergoing apoptosis in vivo are rapidly detected and cleared by phagocytes. Swift recognition and removal of apoptotic cells is important for normal tissue homeostasis and failure in the underlying clearance mechanisms has pathological consequences associated with inflammatory and auto-immune diseases. Cell cultures in vitro usually lack the capacity for removal of non-viable cells because of the absence of phagocytes and, as such, fail to emulate the healthy in vivo micro-environment from which dead cells are absent. While a key objective in cell culture is to maintain viability at maximal levels, cell death is unavoidable and non-viable cells frequently contaminate cultures in significant numbers. Here we show that the presence of apoptotic cells in monoclonal antibody-producing hybridoma cultures has markedly detrimental effects on antibody productivity. Removal of apoptotic hybridoma cells by macrophages at the time of seeding resulted in 100% improved antibody productivity that was, surprisingly to us, most pronounced late on in the cultures. Furthermore, we were able to recapitulate this effect using novel super-paramagnetic Dead-Cert Nanoparticles to remove non-viable cells simply and effectively at culture seeding. These results (1) provide direct evidence that apoptotic cells have a profound influence on their non-phagocytic neighbors in culture and (2) demonstrate the effectiveness of a simple dead-cell removal strategy for improving antibody manufacture in vitro.

Figure 1

Inverse relationship between proportion of non-viable (apoptotic) cells and antibody productivity in hybridoma cultures. 2 × 10⁵ viable 4/C6 hybridoma cells/ml were seeded with different proportions of apoptotic cells (non-viable cells obtained from cultures at the end of plateau phase). Monoclonal antibody (IgG1) productivity was assessed in cell-free supernatants four days later. The inverse correlation was significant (p < 0.005)

Figure 2

Antibody production by hybridoma cells after depletion of non-viable cells by macrophages. Equal numbers of viable 9e10/4 hybridoma cells (2 × 10⁵ /ml) were cultured before (Control, open symbols) or after (Depleted, solid symbols) removal of non-viable cells on day 0 using monolayers of HMDM. In the experiment shown, viability was improved from 57 to 96% by HMDM-mediated depletion of non-viable cells. At the indicated times, cell-free supernatants were assayed for IgG1 content. (A) total IgG1 per ml; (B) IgG1 per cell.

Figure 3

Dead-Cert™ Nanoparticles: simple devices for effective direct removal of non-viable cells in vitro. ImmunoSolv's antibody-based Dead-Cert™ technology (see www.immunosolv.com) has been developed to discriminate non-viable from viable cells in vitro. ImmunoSolv has used antibodies, together with additional discriminatory molecules coupled to the surface of 250 nm super-paramagnetic nanoparticles, to develop a simple, yet highly effective dead-cell removal device, the Dead-Cert™ Nanoparticle. Because of their ability to bind to membrane structures that are not accessible on viable cells-represented by the red symbols in the cartoons-the coated particles are able to bind selectively to apoptotic (dying) as well as necrotic (dead) cells and cell debris. Once bound to non-viable cells and debris, the Dead-Cert™ Nanoparticles can be readily removed with the aid of a simple magnet leaving the ‘untouched’ viable cells purified by negative selection.

Figure 4

Depletion of non-viable hybridoma cells using Dead-Cert™ Nanoparticles. Hybridoma cells (9e10/18) taken from the end of plateau phase of culture were subjected to a single magnetic separation using Dead-Cert™ Nanoparticles. (A) Light micrographs of untreated (Control) and separated (Depleted) cells exposed to trypan blue to label dead cells. (B) Quantitative analyses of untreated and separated cell populations. Cells were counted in Neubauer haemocytometer chambers following exposure to trypan blue.

Figure 5

Antibody production by hybridoma cells after depletion of non-viable cells by Dead-Cert™ Nanoparticles. Equal numbers of viable 9e10/18 hybridoma cells (2 × 10⁵/ml) were cultured before (Control) or after (Depleted) removal of non-viable cells on day 0 using Dead-Cert™ Nanoparticles. In the experiment shown, viability was improved from 64% to 95% by Dead-Cert™ Nanoparticle-mediated magnetic depletion of non-viable cells. At the indicated times, cell-free supernatants were assayed for IgG1 content. (A) Total IgG1 per ml; (B) IgG1 per cell.