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AbstractMass transfer in a protein A medium with a silica matrix was analyzed in this study. Using confocal laser scanning microscopy, we monitored the positional and temporal changes in the polyclonal human IgG during batch adsorption. A simple theoretical model based on Fick’s law of diffusion and Langmuir adsorption was used to numerically approximate the external and intraparticle mass-transfer properties of IgG in single particles. The diffusivities found in this study were validated by comparing with the values reported in other studies and also by using numerical calculation to reproduce the experimentally obtained breakthrough curve.

Virus-like particles (VLPs) are composed of one or several recombinant viral surface proteins that spontaneously assemble into particulate structures without the incorporation of virus DNA or RNA. The baculovirus-insect cell system has been used extensively for the production of recombinant virus proteins including VLPs. While the baculovirus-insect cell system directs the transient expression of recombinant proteins in a batch culture, stably transformed insect cells allow constitutive production. In our recent study, a secretory form of Japanese encephalitis (JE) VLPs was successfully produced by Trichoplusia niBTI-TN-5B1-4 (High Five) cells engineered to coexpress the JE virus (JEV) premembrane (prM) and envelope (E) proteins. A higher yield of E protein was attained with recombinant High Five cells than with the baculovirus-insect cell system. This study demonstrated that recombinant insect cells offer a promising approach to the high-level production of VLPs for use as vaccines and diagnostic antigens.

The production of β-galactosidase by Sf9 cells infected with recombinant Autographa californicanucleopolyhedrovirus (AcNPV) was investigated in shake-flask culture using two serum-free basal media: Grace's medium and TNM-FH (Grace's medium supplemented with lactalbumin hydrolysate and yeast extract). At the time of infection, cells grown in serum-supplemented TNM-FH were transferred into fresh basal media without adaptation. The absence of serum depressed the β-galactosidase yield considerably in Grace's medium, but to a much lesser extent in TNM-FH, where it reached around 2/3 of the level obtained in TNM-FH supplemented with 10% fetal bovine serum (FBS). While both lactalbumin hydrolysate and yeast extract promoted β-galactosidase production, their removal by medium replacement on post-infection day 1 gave a β-galactosidase yield nearly equal to that obtained in their continuous presence. Supplementation of basal media with phosphatidic acid (PA) from egg yolk lecithin, which has been shown to enhance cell growth and recombinant protein production in serum-free culture of Chinese hamster ovary (CHO) cells, was also effective in increasing β-galactosidase yield. Elevating the multiplicity of infection (MOI) from 2 to 10 plaque-forming units per cell (pfu/cell) also resulted in an increase in product yield. These results provide information important to the development of cost-effective serum-free culture technology for use in large-scale production of recombinant proteins by the baculovirus-insect cell system.

Long-term cultivation of anchorage-independent animal cells immobilized within porous biomass support particles (BSPs) using a gas-stirred circulating bed fermentor (CBF) was investigated. Inoculation of mouse myeloma MPC-11 (ATCC CCL 167) cells into reticulated polyvinyl formal resin BSPs (3 × 3 × 3 mm; mean pore diameter, 60 µm; porosity, 0.88) and the repeated batch culture of inoculated cells were performed under gentle circulation of BSPs, induced by sparging air from the base of the fermentor. The glucose uptake rate of cells decreased in the initial period just after the start of circulation, since a relatively large number of cells leaked from the BSPs. After that period, the uptake rate gradually increased and the leakage of cells diminished. In the meantime, when inoculated cells were incubated statically by introducing air into the upper part of the fermentor for the initial several days before circulating the BSPs, glucose consumption became very rapid and cell density in the BSPs reached at least 107 cells/cm3 BSP. Thus, a long-term cultivation without significant leakage of cells and with high cell density in BSPs was successfully achieved in the CBF-BSP system.

The specific oxygen uptake rate of hybridoma cells immobilized in calcium alginate gel particles was measured, and the observed data was compared with those of non-immobilized cells. The uptake rate of the immobilized cells coincided with that of the non-immobilized hybridoma cells just after immobilization, but increased with cell growth. On the other hand, the cellular glucose consumption rate decreased slightly during the experiments. The increased oxygen uptake rate by immobilized cells was closely related to the formation of cell colonies in the gel particles.

Continuous production of monoclonal antibody was achieved in serum-free medium by hybridoma cells immobilized by calcium alginate. The cells were cultivated in an expanded bed fermentor under mild flow conditions which reduced destruction of the immobilized gel particles. Monoclonal antibody was produced continuously for more than 40 days.

Growth and death of anchorage-independent animal cells entrapped within porous biomass support particles (BSPs) in static or shake-flask cultures were evaluated by comparison of enzyme activity with non-immobilized cells grown under static culture using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and release of lactate dehydrogenase into the culture medium. Mouse myeloma MPC-11 (ATCC CCL 167) cells inoculated within porous polyvinyl formal resin BSPs (3 × 3 × 3 or 2 × 2 × 2 mm; mean pore diameter, 60 µ ) grew exponentially at a specific growth rate comparable to that of non-immobilized cells in the initial period of incubation. Entrapped cells then reached the stationary phase with a cell density over 107 cells/cm3 BSP. The death rate of entrapped cells increased in response to the rise in viable cell density in the BSPs. Observation of viable cell distribution within the BSPs using MTT staining indicated that the cells concentrated within a thin outer shell of the BSPs with time. After the immobilized cells reached the stationary phase, penetration of cells into the outer shell ceased and heterogeneous distribution of cell density occurred in the viable cell layer in the shake-flask culture.

Immobilisation of anchorage-independent animal cells using Biomas Support Particles (BSPs) was investigated. Mouse myeloma MPC-11 cells were physically entrapped in three-dimensional reticulated polyvinyl formal (PVF) resin PSPs (3×3×3 mm) with matrices of relatively small pores (30–100 µm) by filtering medium containing cells or incubating in a shake flask for inoculation. Physically entrapped cells became immobilised in the BSPs by forming aggregates within the matrices of the reticulated PVF resin, and cell density in the BSPs reached at least 107 cells/cm3 BSP. Immobilised cells in the BSPs were successfully cultivated in static and/or shake-flask cultures with regular replacement of medium for a long period.