Biological drug products from cells, such as DNA vaccines and proteins, have been mass-produced by recombinant gene technology. These drugs often contain lipopolysaccharides (LPSs), which originate from the cell wall of the host cells and other contaminants. Residual LPSs must be removed from biopharmaceutical product solutions used for injection because they cause pyrogenic and shock reactions in mammals on intravenous injection, even in nanogram quantities.
To date, cationic adsorbents have been used for removing LPSs from bioproduct solutions. However, it has been reported that cationic adsorbents cannot selectively remove LPSs from acidic substances, such as DNA, because of the high adsorption of both LPSs and DNA by an ionic interactions. We have reported development of water-insoluble copolymer particles of γ-CyD and urethane (1,6-hexamethylene diisocyanate, HMDI) as a novel LPS adsorbent and compared their adsorption capacity with γ-CyD/CMO particles.1 HMDI is a more hydrophobic cross-linker than CMO, and it was expected to enable wide control of the CyD content during the preparation of water-insoluble γ-CyD/HMDI adsorbents. This article describes the effect of γ-CyD content within the adsorbent on the LPS adsorption capacity and the effect of the buffer’s conditions (ionic strength and pH) on selective removal of LPSs from DNA solutions. Finally, we compare the LPS removal performance of γ-CyD/HMDI with that of the cationic and hydrophobic LPS adsorbents.
Selective removal of LPSs from various bioproduct solutions was investigated under physiological conditions (pH 7.0, l = 0.16). Various bioproduct solutions, which were naturally contaminated with LPSs at concentrations from 15 to 112 EU ml-1, were used as samples. γ-CyD/HMDI-20/80 particles was able to remove natural LPSs from each compound to a level below 1 EU ml-1, and a high recovery (99%) of compound was obtained with each sample after removal of LPSs.
The results indicate that the adsorption method using γ-CyD/HMDI particles can be used to reduce LPS contamination from crude DNA vaccine materials. This batch method shows good recovery of DNA (recovery > 99%) and sufficient LPS removal activity (residual concentration of LPS >1 EU ml-1) under physiological conditions. The high LPS selectivity of the particles with γ-CyD cavities is possibly due to (i) the effect of including the hydrophobic chain of LPSs into the cavity and (ii) the size exclusion effect on DNA molecules.
Figure1Chemical structures of cross-linked γ-CyD particles and a schematic diagram of a structure of lipid A of LPS and its inclusion behavior into the cavity of γ-CyD.
In recent years, the immobilization of bioactive molecules on solid supports has given rise to a wide range of academic and industrial applications, e.g., immobilized enzyme reactors for proteomics. Proteases are the predominant industrial enzymes, accounting for about 60% of total worldwide sales. Immobilized enzymes are reusable and easily removed from the digestion medium. Several reports have demonstrated proteolytic performances using enzymes immobilized on various supports such as magnetic nanoparticles, nanodiamonds, mesoporous activated carbon, mesoporous silica, and glass beads.
GO nanosheets (GONSs) have attracted attention as potentially useful materials for bioapplications because of their unique properties such as planar morphology, chemically active surface area, and stability toward corrosive media. The surface of the GONS is covered with a variety of functional groups including carboxy, epoxy, and hydroxy groups, which may be easily activated for conjugation of biological molecules. Moreover, GONSs have high surface ratios (2600 m2g−1) and also high dispersibility in liquid media because of their oxidized, hydrophilic surfaces.
We have developed a novel enzyme-immobilized GONS using a one-step and natural reaction.2 Trypsin was successfully immobilized on the GONS by diimide-activated amidation. The amount of trypsin immobilized on the GONS (1.9 mg•mg−1) was higher than those immobilized on nanoparticle supports. A large amount of trypsin was immobilized on the GONS surface as a result of its high surface ratio and its large content of functional (carboxy) groups. Furthermore, compared with free trypsin, trypsin GONS showed higher stability to temperature and ethanol. This high stability of trypsin GONS is a result of stabilization of weak intermolecular forces and prevention of enzyme autolysis when trypsin GONS is sufficiently dispersed in solution. The whole amidation process was conducted within only 60 min, which is easy to scale up at a low cost. These results are expected to open up new applications of GONSs in proteomics.
Figure2Top: A schematic representation of immobilization of trypsin on magnetic graphene oxide. Bottom: AFM images of trypsin-immobilized GO nanosheet, which were prepared by dropping a nanosheet suspension onto a mica surface.
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