Affinity Chromatography Media
for Endotoxin Removal

CellufineTMET clean

Introduction

The Cellufine™ ET clean is poly(ε-lysine) immobilized Cellufine™ (cellulose spherical beads). The beads bind and remove endotoxin from your sample solution. The poly(ε-lysine) is a microbial poly(amino acid) that consist of 25-35 lysine residues produced by Streptomyces albulus. The poly(ε-lysine) as ligand and the cellulose beads act as matrix ands are products of JNC Corporation.

The Cellufine™ ET clean endotoxin removing beads were developed jointly by Kumamoto University and Chisso. The poly(ε-lysine) was immobilized onto chloromethyloxirane-activated cellulose beads. The beads are a stable affinity beads that are resistant against the cleanup solutions, which include 0.2 M sodium hydroxide and 2 M sodium

The Cellufine™ ET clean can remove endotoxin from a cellular product solution at physiological pH, ionic strength of μ = 0.02-1.0, and 0° -25C°.

Electron micrograph of Cellufine™ ETclean-S beads.
Electron micrograph of Cellufine™ ETclean-S beads.

Partial Structure

Partial Structure

Characteristics

Name Supplied Wet Bead Diameter Pore Size*
Cellufine™ ET clean S a slurry in 20 % ethanol ca. 40-130 μm Mlim 2000
Cellufine™ ET clean L a slurry in 20 % ethanol ca. 40-130 μm >Mlim 2x106

*The pore size (molecular weight exclution; Mlim) of the beads was estimated from calibration curves obtained by size exclusion chromatography. Pullulan and maltose were used for the Mlim determination.

Selective adsorption of endotoxin (LPS) from a bovine serum albumin (BSA) solution by Cellufine ET clean beads.

Selective adsorption of endotoxin (LPS) from a bovine serum albumin (BSA) solution by Cellufine ET clean beads.

Selective adsorption of endotoxin was determined using a batchwise method with 0.2 g of the wet beads and 2 ml of a sample solution (BSA: 500 μg/ml, E. coli O111: B4 LPS: 100 ng/ml, pH 7.0, ionic strength of μ = 0.05-0.8 ).

Selective removal of endotoxin from a protein solution by Cellufine™ ET clean beads.

Sample Solution Cellufine™ ET clean S Cellufine™ ET clean L
Compound(pI) Concentration of endotoxin before treatment(pg/ml) (μ = 0.05, pH 7.0) (μ = 0.4, pH 7.0)
Concentration of endotoxin after treatment(pg/ml) Recovery of protein after treatment(%) Concentration of endotoxin after treatment(pg/ml) Recovery of protein after treatment(%)
Ovalbumin(4.6) 28,000 81 99 <10 95
BSA(4.9) 32,000 45 99 <10 97
Myoglobin(6.8) 4,500 18 99 <10 98
γ-globulin(7.4) 5,600 20 99 <10 97
Cytochrome C(10.6) 1,500 15 99 <10 98

The removal of endotoxin was determined by a batchwise method with 0.3 ml of wet adsorbent and 2 ml of a protein solution (1 mg/ml) containing natural endotoxin.

Application Data

ET removal Exp.
BSA / ETclean L

Column chromatography

Column size
1 X 1.1 cm (I.D.) (1.1ml)
Flow rate
0.17 ml / min (10cm / h)
Buffer
50 mM PB, pH 7 + 0.15 mol NaCl aq

Assay

- Protein Abs. at 280 nm
- ET LAL rate assay

- Injection sample (150 ml)
- BSA 1 mg/ml ET 100 EU/ml

Removal of the endotoxin from Albumin.
Removal of the endotoxin from Albumin.

Lysozyme / ET clean L

Column chromatography

Column size
10 x 0.9 cm (I.D.) (9.6 ml)
Flow rate
0.5 ml / min (47 cm / h)
Buffer
1 mM Tris-HCl, pH 7.3
Gradient
0 → 1.0 mol / l NaCl aq.

Assay

- Protein Abs. at 280 nm
- ET LAL rate assay

- Injection sample (1ml) : 14 mg / ml

Removal of the endotoxin from Lysozyme.
Removal of the endotoxin from Lysozyme.

Insulin chain A / ET clean L
- Injection sample (1ml) : 13 mg / ml, 309 EU / ml

Insulin chain A / ET clean L

Tranceferrin / ET clean L
- Injection sample (1ml) : 13 mg / ml, 2982 EU / ml

Tranceferrin / ET clean L

References

1) M. Sakata, M. Todokoro, C. Hirayama, American Biotechnol. Lab., 20 (2002) 36.
2) M. Todokoro, M. Sakata, S. Matama, M. Kunitake, J. Ohkuma, C. Hirayama, J. Liq. Chrom. & Rel. Technol., 25 (2002) 601.

Development of the grade

Development of the grade according to demand of a customer (inquiry)

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