Tomoko Hirasaki

Permeation mechanism of DNA molecules in solution through cuprammonium regenerated cellulose hollow fiber (BMMtm)

Abstract We tried to clarify the membrane permeation mechanism of biopolymer DNA molecule in solution through cuprammonium regenerated cellulose hollow fiber (BMM tm ) from the dependence of the permeability of the DNA molecule on the molecular weight (MW), the transmembrane pressure ( ΔP ), the total challenge dose, the original concentration of DNA and on the conformation of DNA and from the observation of the shape of the DNA molecule remaining in the membrane wall. The shape of the DNA molecule was observed using transmission electron microscopy (TEM). The permeability of DNA molecules decreased with an increase in the MW of the DNA molecule. The MW of the molecule which showed a permeability of more than 0.9 was 1 × 10 6 for the protein with global configuration and 1 × 10 8 for DNA. The linear protein of the blood coagulation factor VIII combined with von Willebrand factor (F-VIII with vWF) with MW of 2 × 10 7 showed a permeability similar to that of DNA rather than that of the global protein. When ΔP decreased, the permeability of DNA decreased. Electron microscope observation showed that the DNA molecules were elongated by the shear stress originated in the flow of the solution in pores. We can conclude that: (1) Protein and DNA permeate through BMM mainly based on sieving effects. (2) The molecules of DNA and F-VIII with vWF are considered to deform into a string shape along the stream line. The chemical structure of a molecule and the shear stress of filtration govern its deformability. The deformation of the molecule contributes to the permeability through BMM. (3) The sieving effect in working on the permeation of molecules should take into account their deformability in addition to their geometrical size.

Sieving study of chromatin and histone-DNA complex by porous hollow fiber membranes

Abstract Permeation of chromatin isolated from human HeLa S3 cells and histone-DNA complex through regenerated cellulose hollow fibers was investigated as a model study of DNA removal for drug manufacturing using membrane technology. It was found that the permeation of histone-DNA and chromatin through the membranes having a mean pore diameter of 15 nm was substantially lower than the permeation of free DNA which did not complex with proteins based on the concentration determination of the feed and permeate solutions using UV spectroscopy. Direct observation to determine the existence of chromatin and histone-DNA complex in the permeate solution was also performed using atomic force microscopic imaging. No chromatin and chromatin-like structures were found in the examination of 10 areas of 1.0×1.0 μm on the mica which had been adsorbed from the solution permeated through the membranes. Thus, the membranes having a mean pore diameter of 15 nm most likely reject the chromatin and histone-DNA complex that are observed to have an apparent width of 25–30 nm based on atomic force microscopic imaging.

Virus Containment Continuous Culture of Animal Cells

Human-human hybridoma HB4C5 cells were cultured in a high-density continuous culture system SHC-1 (SHIMADZU) equipped with a 500 ml culture vessel at the density of 2 × 107 cells/ml. Cultured medium were filtrated by the BMM hollow fiber module which can eliminate virus particles. This module was able to filtrate over 20 L of cultured medium over 30 days without decrease in the recovering rate of the cultured medium. The high density continuous culture system installed with the BMM hollow fiber can be used for virus containment in the vessel and producing virus-free products by continuous culture.

Removal of DNA in Cell Cultured Fluid Using BMMTM

We showed that the large amount of cellular DNA contaminated in cell cultured medium. For removal and/or reduction of contaminated DNA in bio-drugs, we want to show the applicability of the cuprammonium regenerated cellulose hollow fiber (BMMTM) virus removal filter. In cell culture step, that is, upstream manufacturing process of bio-drugs, the removability of DNA and the permeability of protein produced by cell using BMM were checked. Cell cultured fluid by hybridomas was prepared. Cell cultured fluid was filtered using BMM with mean pore size of 15nm and 35nm (BMM15 and BMM35). The amount of monoclonal antibodies (MAbs) (i.e., IgG and IgM) produced by cell were determined by ELISA. The removability of DNA by filtration was about 2 logs rejection and decreased with the increase of cell culture day. It may be caused by the differences of both dispersion state of DNA and fragmentation of DNA during culture. The permeabilities of IgG and IgM kept higher level than that of DNA in cultured fluid. DNA in protein solution was easily reduced comparing with naked DNA using BMM.