A method for depletion or removal of endotoxin from an endotoxin-containing source or potentially endotoxin-containing source
A technology of endotoxin, source, applied in the field of fractionation
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Embodiment 1
[0112] Experimental controls were prepared to provide a baseline for recording the relative effectiveness of the invention.
[0113] 10 mL of a clarified harvest of E. coli cell culture containing phage T4 was partially purified by hydrophobic interaction chromatography (HIC). With 50mM Tris, 1mM CaCl 2 , 0.5mM MgCl 2 , 20mM NaCl, 10% glycerol, pH7.7, and the final conductivity was 7.42mS / cm to elute HIC to obtain the phage elution peak. 100 μL CIMmac Qa (strong anion exchange) monolith (BIA Separations) was washed with 50 mM Tris, 1 mM CaCl 2 , 0.5mM MgCl 2 , 20mM NaCl, 10% glycerol, pH7.7 balance. Load the diluted phage onto the QA monolithic column. Wash the monolithic column with 60 bed volumes of equilibration buffer, then elute with a 50 bed volume (5 mL) linear gradient to 50 mM Tris, 1 mM CaCl 2 , 0.5mM MgCl 2 , 2M NaCl, 10% glycerol, pH 7.7. The column was cleaned and disinfected with 1M sodium hydroxide and 2M sodium chloride. Fractions were collected throug...
Embodiment 2
[0114] Embodiment 2 (comparison)
[0115] Endotoxin reduction experiments were performed initially with hydrophobic interaction chromatography (HIC) followed by IDA-Fe, a negatively charged chelating ligand (CIM IDA, BIA Separations). HIC was performed on T4 phage harvest as described in Example 1, but on a larger scale, with 160 mL of lysate loaded onto an 8 mL HIC monolithic column. The endotoxin content of 31mL eluted fraction was 138,167EU / mL. The fraction was treated with 20nM KH 2 PO 4 , 10% glycerol, pH 7.0 diluted to a final conductivity of 6.7mS / cm. A 340 μL IDA monolithic column was prepared by washing with 20 Cv deionized water; then 20 CV 100 mM acetic acid, pH 3.0; 20 CV 200 mM ferric chloride, then 20 CV 50 mM acetic acid, 2.0 M sodium chloride, pH 4.5; 20 Cv deionized water. The column was then equilibrated to 20 mM potassium phosphate, 10% glycerol, pH 7.0. Dilute 2(2) mL of the fraction from HIC with 40 mL of mobile phase A to a conductivity of 6.7 mS / c...
Embodiment 3
[0117] Experiments show that the experimental monolithic column produced by BIASeparations immobilized ferric ions on the solid surface of tris(2-aminoethyl)amine (TREN) achieves a higher yield compared with strong anion exchangers (CIMmultus QA, BIA Separations). Good endotoxin reduction.
[0118] HIC was performed on T4 phage harvests as described in Example 2. Endotoxin detection showed that the endotoxin content of the phage fraction was 138167EU / ml. Dilute 2 mL of the fraction from HIC with 40 mL of mobile phase A to give a final conductivity of 9.3 mS / cm. A 1 ml TREN monolithic column was prepared as described in Example 2 by running reagents on it at a flow rate of 5 CV / min. The column was equilibrated with 20 mM potassium phosphate (pH 7.0). 40 mL of the diluted sample was loaded, and the column was washed with 15 CV of 20 mM potassium phosphate, pH 7.0. The column was eluted with a 20CV linear gradient with an endpoint of 500 mM potassium phosphate, pH 7.0. Fract...
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