Masayo Sakata

Chromatographic removal of endotoxin from protein solutions by polymer particles.

Endotoxins, constituents of cell walls of gram-negative bacteria, are potential contaminants of the protein solutions originating from biological products. Such contaminants have to be removed from solutions used for intravenous administration, because of their potent biological activities causing pyrogenic reactions. Separation methods used for decontamination of water, such as ultrafiltration, have little effect on endotoxin levels in protein solutions. To remove endotoxin from a solution of high-molecular-mass compounds, such as proteins, the adsorption method has proven to be most effective. In this review, we first introduce endotoxin-specific properties in an aqueous solution, and then provide various methods of chromatographic separation of endotoxins from cellular products using polymer adsorbents. We also provide the design of novel endotoxin-specific polymer adsorbents.

Formation of the ‘nanotube’ structure of β-cyclodextrin on Au(III) surfaces induced by potential controlled adsorption

Abstract The self-organization of β-cyclodextrin (β-CyD) into a ‘nanotube’ structure, similar to that of CyD-polyrotaxane, was found to be induced by potential controlled adsorption on Au(III) surfaces in sodium perchlorate solution without a threaded polymer. In-situ scanning tunneling microscopy (STM) revealed that the cavities of β-CyD faced sideward not upward in the tubes. This ordered structure can form only under conditions where the potential is controlled (−0.45 V to −0.25 V vs. SCE). β-CyD molecules were in a disordered state on bare Au(III) surfaces without potential control (+0.00 V vs. SCE). In addition, the desorption of β-CyD from Au surfaces was observed at a negative potential of less than −0.60 V. In the range −0.45 to −0.25 V, β-CyD molecules formed ordered arrays on Au(111) surfaces. Furthermore, the discontinuity of potential control led to disordered phases and the destruction of the ‘tube’ structure. This indicates that by controlling the electrode potential a delicate balance of various interactions can be achieved, resulting in the self-organization of molecules on the surface.

Preparation of poly(ε-lysine) adsorbents and application to selective removal of lipopolysaccharides

To remove endotoxins (lipopolysaccharides; LPS) from cell products used as drugs, water-insoluble poly(e-lysine) (PL) particles were prepared by cross-linking with PL originating from Streptomyces albulus and chloromethyloxirane (CMO). The apparent pKa (pKa,app) and the anion-exchange capacity of the particles were easily adjusted by changing the PL ratio and the CMO ratio. The higher the pKa,app, the greater the LPS-adsorption capacity of the particles. On the other hand, when the PL ratio (in the particles) increased to 75 unit-mol% or higher, the adsorption of bovine serum albumin by the particles also increased, but decreased with increasing ionic strength of the buffer to μ=0.2 or higher. The adsorption of γ-globulin increased with decreasing PL ratio to 65 unit-mol% or lower. As a result, when the PL ratio was 70 unit-mol% and the pKa,app was 6.7, the PL/CMO particles selectively removed LPS from various protein solutions that were naturally contaminated with LPS, at pH 6.0 and μ=0.05.

In situ observation of coronene epitaxial adlayers on Au(111) surfaces prepared by the transfer of Langmuir films

Abstract A highly ordered epitaxial adlayer of coronene on an Au(111) surface was prepared by a ‘wet process’ technique, which consisted of the simple transfer of Langmuir films. The structure and dependence on potential of the adlayer were then investigated by in situ scanning tunneling microscopy (STM). The adlayer possessed a (4×4)-Au(111) superlattice structure, and each coronene molecule was visualized as an individual hexagon on high-resolution STM images. The multilayer portion was present without any potential control, probably due to the excess generated by the transfer. The adsorption behavior of coronene, including the formation of the multilayers, showed dependence on the potential. A flawless adlayer without the presence of any multilayers was achieved by the application of a negative potential, in which the multilayer–substrate interaction was adequately weakened.

Cross-linked N,N-dimethylaminopropylacrylamide spherical particles for selective removal of endotoxin

Abstract Spherical polymer adsorbents for endotoxin adsorption were prepared by suspension copolymerization of N,N-dimethylaminopropylacrylamide (DMAPAA) with N-allylacrylamide (AAA). The amino-group contents and the pore size of the adsorbents were easily adjusted by changing the monomer ratio and diluent ratio. The more amino groups are introduced, the larger is the endotoxin-adsorbing capacity of the adsorbent, and the smaller the pore size (molecular mass exclusion, Mlim) of the adsorbent, the less acidic proteins such as bovine serum albumin are adsorbed. When Mlim was smaller than 300 (as the molecular mass of polysaccharide) and the amino group content was 4.5 mequiv. /g, the DMAPAA-AAA adsorbent showed a high endotoxin-removing activity at an ionic strength of μ = 0.05-0.4 and pH 5–9. The adsorbent was also able to remove endotoxin from a protein solution, naturally contaminated with endotoxin, at μ = 0.05 without affecting the recovery of the protein. The adsorbent can be completely regenerated by washing with 0.2 M sodium hydroxide followed by 2.0 M sodium chloride.

Selective assay for endotoxin using poly(ε-lysine)-immobilized Cellufine and Limulus amoebocyte lysate (LAL)

We developed a selective endotoxin (lipopolysaccharide; LPS) assay using poly(epsilon-lysine)-immobilized cellulose beads (PL-Cellufine) and Limulus amoebocyte lysate (LAL). First, LPS was selectively adsorbed on the beads in a solution containing various LAL-inhibiting or LAL-enhancing compounds (e.g., amino acids, enzymes) and the LPS adsorbed on the beads was separated from the compounds by centrifugation. Second, the LPS adsorbed on the beads directly reacted with the LAL reagent, and the LPS concentration was determined by a turbidimetric time assay. The accuracy of the adsorption method with PL-Cellufine was high compared with that of a common solution method. Apparent recovery of LPS from compound solution was 88-120%.

Self-organization of α-cyclodextrin on Au(111) surfaces induced by potential controlled adsorption

Abstract Potential-induced self-organization of α-cyclodextrin (CyD) on Au(111) surfaces in sodium perchlorate has been investigated by using in-situ scanning tunneling microscopy (STM). Near the open circuit potential (approximately +0.0 V vs. SCE), the CyD molecule adsorbs randomly on bare Au(111) surfaces and the desorption of CyD from Au surfaces is observed at negative potentials less than −0.40 V. On the other hand, it was found that the CyD molecules formed an ordered array with a cylindrical structure on the Au(111) surface in the potential region from −0.20 to −0.15 V. The cylindrical structure in which the cavities of CyD face sideways proved that the structure of the adlayer is formed not by simple epitaxy but by self-organization of the molecules.

2D-supramolecular arrangements of dibenzo-18-crown-6-ether and its inclusion complex with potassium ion by potential controlled adsorption

The construction of self-organized adlayers of dibenzo-18-crown-6 and its inclusion complex with potassium ion on a Au(111) surface was independently achieved by potential-controlled adsorption. Highly-ordered adlayers both for the metal-free state and the complex were visualized by in-situ STM with sub-molecular visualization. The potassium ions in the complex were also clearly visualized.

Pore-size controlled and poly(ε-lysine)-immobilized cellulose spherical particles for removal of lipopolysaccharides

Poly(ϵ-lysine) was covalently immobilized onto cellulose spherical particles and used for selective adsorption of pyrogenic lipopolysaccharides (LPS) from protein solutions. The resulting poly(ϵ-lysine)-immobilized cellulose particles (PL-cellulose), which had diameters of 44 to 105 μm and matrixs pore-sizes of 2 × 103, 1 × 104, and >2 × 106 as molecular mass exclusions (Mlim), were used as adsorbents. The adsorption of LPS and protein to the adsorbent were determined using a batchwise method. The larger the pore size (Mlim) of the adsorbent, the larger is the LPS-adsorbing activity of the adsorbent. The apparent dissociation constant between the LPS (E. coli O111:B4) and the adsorbent decreased from 3.8 × 10−10 to 1.1 × 10−11 M with an increase in the Mlim from 2 × 103 to >2 × 106 at an ionic strength of μ = 0.05 and a pH of 7.0. On the other hand, the adsorbing activity of bovine serum albumin also increased with the increasing Mlim of the adsorbent, but sharply decreased with increasing ionic strength...

The removal of endotoxins from protein solutions using column packings with aminated poly (γ-methyl L-glutamate) spheres

SummaryA method is described for the selective removal of endotoxins from various protein solutions using columns packed with aminated poly (γ-methyl L-glutamate) (PMLG-NH2) spheres. The PMLG-NH2 adsorbents showed a high adsorbing activity for endotoxins which had an ionic strength of μ=0.05–1.0 and pH 5.0–9.0. The endotoxin-adsorbing capacity per millitre of the wet adsorbent increased from 0.40 to 1.35 mg (E. coli O111: B4 LPS) at μ=0.2 and pH 7.0 while the aminogroup content of the adsorbent increased from 0.8 to 3.5 meq g−1. The PMLG-NH2-3.5 has an amino-group content of 3.5 meq g−1. This column packing selectively adsorbed endotoxins, without loss of the protein, from a γ-globulin or cytochromec solution which contained endotoxins at μ=0.05 and pH 7.0. On the other hand, when bovine serum albumin (BSA) was present in solution with endotoxins, both the endotoxins and the BSA were adsorbed by the column. The BSA-adsorbing activity increased with increasing amino-group content of the adsorbent. However, this undesirable adsorption was suppressed with increasing ionic strength of the buffer. As a result, when the packing which had an amino-group content of 1.5 meg g−1 was used in conditions of μ=0.2 and pH 7.0, the endotoxins were removed from a BSA-containing solution without affecting the recovery of the BSA.