A high-yield and scaleable adenovirus vector production process based on high density perfusion culture of HEK 293 cells as suspended aggregates.

Cells of the human embryonic kidney cell line (HEK 293) were grown as suspended aggregates in stirred vessels and infected with a recombinant adenovirus vector (Ad-TH-GFP). Regular spherical aggregates with the mean diameter less than 300 microm and a viable cell density greater than 5 x 10(6) cells x ml(-1) were readily achieved after 9 day culture in spinner flasks. The HEK 293 cells growing as suspended aggregates could be efficiently infected by Ad-TH-GFP at an MOI of 10 with a prolonging infection time up to 144 hour post-infection (hpi). The time profile of Ad-TH-GFP production was strongly corresponding to the infection process with a virus concentration peak occurred consistently at 144 h after infection. And the infected aggregates essentially maintained spherical in shape, the portion of dissociated cells from the infected aggregates was less than 5% at 144 hpi. Perfusion culture of HEK 293 cells grown as suspended aggregates in a 7.5 L stirred tank bioreactor and infected with Ad-TH-GFP at a density higher than 1 x 10(7) cells x ml(-1) resulted in a similar Ad-TH-GFP production kinetics, but a much higher virus yield approximately at 5.7 x 10(11) GTU ml(-1) at 144 hpi to that of the infected spinner flask cultures. These results demonstrate the feasibility for using suspended cell aggregates as an immobilization system to facilitate perfusion in stirred tank bioreactors, and improve volumetric productivities by eliminating the cell density effect.

[In vitro cytolysis of B-lymphoma cells mediated by an anti-CD3/anti-CD20 bispecific single-chain antibody].

After having successfully constructed and expressed the gene of the anti-CD3/anti-CD20 bispecific single-chain antibody (bscCD3 x CD20), here we analyzed its in vitro bioactivity of mediating the lysis of Ramous human B-lymphoma cells in the presence of T-enriched human peripheral blood lymphocytes (PBL). Obvious opoptosis characters were observed by Annexin V/PI(AV/PI) stained and scanning electron microscope. As evaluated by non-radioactive cytotoxity assay, the bscCD3 x CD20 showed potent bioactivity of mediating human B-lymphoma cells lysis in the presence of T-enriched human PBL. The potency of cytotoxicity depended on the ratios of effect cells to target cells (E:T) used. Further, the antibody showed a dose and time-dependent effect on mediating Ramous cells lysis. The specific lysis reached about 87.3% at an antibody concentration of 5microg/mL and E:T used at 10:1. Clear changes in apoptogenes expression profiles were detected by apoptosis gene array after Ramous cells were treated with the antibody and PBL. Among the upregulated apoptogenes, ATM and P53 showed an increase of 187 times and 15 times respectively, which suggested that ATM-p53 pathway may be the main apoptosis way of Ramous cells induced by T cells in the presence of the bscCD3 x CD20.

[Expression, identification and bioactivity characterization of an anti-CD3/anti-CD20 bispecific single-chain antibody].

The synthetic gene with 1640bp encoding for the anti-CD3/anti-CD20 bispecific single-chain antibody was designed and obtained by SOE (splicing by overlap extension) PCR. The cDNA was cloned into Flp-In expression vector pcDNA5/FRT and transfeced into Flp-In CHO cells to generate a stable expression cell line with a capacity for expressing anti-CD3/anti-CD20 bispecific single-chain antibody at 300 microg/L. The protein, which had a molecular weight of about 70 kD,was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western-blot analysis. Immunofluorescence assay and cellular rosetting showed that it can react specifically on Jurkat (CD3+) and Ramous (CD20+) cells. The lysis of human PBL against CD20-positive lymphoma Ramous cells in the presence of the anti-CD3/anti-CD20 bispecific single-chain antibody can observed by microscope. All these results would lighten the further study of its biological functions in vitro and in vivo.