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introductionThe pharmaceutical products for parenteral administration must be tested for contamination of bacterial endotoxins (LPSs 1 ) because of their potent biologic activities, which can cause pyrogenic and shock reactions. LPS, a constituent of the cell walls of Gram-negative bacteria, is a potential contaminant of protein solutions originating from biologic products. There are two methods for determining LPS. One is the rabbit pyrogen test; the other is the limulus test using Limulus amebocyte lysate (LAL).The gel-clot, 2 turbidimetric, 3 and chromogenic 4 testing methods, methods previously approved by the USA Pharmacopoeia in 1981, 1985, and 1985, respectively, are typically employed in the LAL test. The advantages of the LAL test over the rabbit pyrogen test are higher sensitivity, lower cost, and higher reproducibility. The LAL test is inhibited or enhanced by antibiotics, hormones, heavy metals, amino acids, alkaloids, plasma proteins, enzymes, and electrolytes in the sample solution.5 Dilution, heating and dialysis have been used to overcome these problems, but the results have been unsatisfactory. Minobe et al. attempted to develop a new specific assay method for LPSs using histidine-immobilized Sepharose as an adsorbent.6,7 LPSs adsorbed by the adsorbent were separated from the compounds (e.g., amino acids, enzymes) in solution by centrifugation, and the adsorbed LPSs can react directly with the LAL. The great disadvantage of this type of adsorbent is the low adsorption activity of the LPSs in physiologic solution (neutral pH and ionic strength (μ) = 0.1 -0.2).In this study, we developed a novel assay of LPSs with a new adsorption method using a turbidimetric time assay system. We previously found that poly(ε-lysine)-immobilized cellulose beads (PL-Cellufine) can selectively adsorb LPSs in bioproducts over a wide range of ionic strengths (μ) from 0.05 -0.8 8-10 at neutral pH. On the other hand, a simple turbidimetric time assay system has been developed for the quantitative assaying of LPS; an automated apparatus is available commercially. 11In this paper, we describe the LPS-adsorbing activities of PL-Cellufine. Then, using the turbidimetric time assay, we describe the optimum conditions for the LPS assay using the PL-Cellufine as adsorbent. We also compare the apparent quantity of LPS provided by the adsorption method with the apparent quantity of LPS provided by a normal method. experimental Reagents and materialsPurified LPS (Escherichia coli UKT-B and O111:B4) and Limulus ES-II test Wako (Limulus amebocyte lysate) were purchased from Wako Pure Chemicals (Osaka, Japan). Poly(ε-lysine)-immobilized cellulose beads (PL-Cellufine) [8][9][10] was obtained from Chisso Co. Ltd. (Tokyo, Japan). PL (Mw, 4500; pKa, 7.6; Chisso) was produced by Streptomyces albulus. 12 DEAE-Sepharose CL-6B was purchased from GE Healthcare Bio-Sciences Corp. (USA). Ovalbumin (from egg), bovine serum albumin (BSA), γ-globulin (from human serum), lysozyme (from horse heart), and vitamin K3 were purchased from Wako. Myoglobi...

SummaryTo obtain fundamental information about the surface-environment effect of adsorbent on the selective adsorption of endotoxin (hpopolysaccharide; LPS) from a protein solution, crosslinked poly(c-lysine)(PL) and aminated poly(7-methyl L-glutamate) (PM LG-NH 2) spherical adsorbents were prepared. The apparent pKa (pKa,app) of the PL and PMLG-NH 2 adsorbents was adjusted by controlling the ratio of poly(c-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, ix, 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 xl0 10 to 1.0 xl0 l~ Although PMLG-NH 2 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 ix = 0.05. The residual LPS concentration in each sample was > 100 pg mL 1, and recovery of the protein was >96%.