Masami Todokoro

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.

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%.

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...

Effect of cationic polymer adsorbent pKa on the selective removal of endotoxin from an albumin solution

SummaryTo obtain fundamental information about the surface-environment effect of adsorbent on the selective adsorption of endotoxin (lipopolysaccharide; LPS) from a protein solution, cross-linked poly(ε-lysine)(PL) and aminated poly(γ-methyll-glutamate) (PMLG-NH2) spherical adsorbents were prepared. The apparent pKa (pKa,app) of the PL and PMLG-NH2 adsorbents was adjusted by controlling the ratio of poly(ε-lysine), in cross-linking, and the amination conditions (time and ratio of diaminoethane in the particles), respectively. When adsorption of LPS and acidic protein, e.g. bovine serum albumin (BSA), by the particles was determined by a batchwise method at pH 7.0 and an ionic strength, μ, of 0.05, they were found to depend strongly on pKa,app but not necessarily on the amino-group content. When pKa,app was increased from 6.8 to 8.2, the LPS-adsorbing capacity was increased from 1.0 to 2.7 mg mL−1 wet adsorbent and the apparent dissociation constant for adsorbent and LPS decreased from 3.7×10−10 to 1.0×10−10m. Although PMLG-NH2 with the highest pKa,app, 8.2, had the highest LPS-adsorbing activity, it also adsorbed BSA. The BSA-adsorbing activity was sharply reduced when pKa,app was reduced to 6.8 or lower. As a result, the cross-linked PL adsorbent of pKa,app 6.8 had the highest LPS selectivity. The adsorbent could reduce levels of natural LPS associated with acidic protein (ovalbumin or BSA) at pH 7.0 and μ=0.05. The residual LPS concentration in each sample was >100 pg mL−1, and recovery of the protein was >96%.

γ-Cyclodextrin-polyurethane copolymer adsorbent for selective removal of endotoxin from DNA solution

Copolymer particles for removal of endotoxins (lipopolysaccharides, LPSs) were prepared by suspension copolymerization of γ-cyclodextrin (CyD) and 1,6-hexamethylenediisocyanate. The LPS-removing activity of the copolymer particles was compared with that of poly(ε-lysine)-immobilized Cellufine (cationic adsorbent) or polystyrene particles (hydrophobic adsorbent) by a batch method. When DNA was present in solution with LPSs under physiological conditions (pH 6.0, ionic strength of μ = 0.05-0.8), LPS-removing activity of the cationic or hydrophobic adsorbent was unsatisfactory because both the DNA and the LPSs were adsorbed onto each adsorbent. By contrast, the copolymer particles with γ-CyD cavity (CyD content: 14-20 mol%) could selectively remove LPSs from a DNA solution (50 μg ml(-1), pH 6.0, and μ = 0.05-0.2) containing LPSs (15 EU ml(-1)) without the adsorption of DNA. The residual concentration of LPSs in the treated DNA solution was below 0.1 EU ml(-1), and the recovery of DNA was 99%.