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°.

SEM photo of Cellufine ET clean S
Electron micrograph of Cellufine™ ETclean-S beads.

Partial Structure

Ligand structure of Cellufine ET clean S

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.

Removal of endotoxin from proteins with Cellufine ET clean S

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

Endotoxin removal from BSA with Cellufine ET clean L
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 endotoxin from egg white lysozyme with Cellufine ET clean L
Removal of the endotoxin from Lysozyme.

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

Removal of endotoxin from insulin α chain with Cellufine ET clean L

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

Endotoxin removal from transferrin with Cellufine 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.

Custom Resin Development Services

JNC offers a custom resin development service for optimization of current products to meet stringent or enhanced performance working directly with large and small customers. In addition, JNC also offers custom resin development services to support new applications of JNC resins.

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