Materials and methods for removing endotoxins from protein preparations

A protein preparation and endotoxin technology, applied in the field of materials and methods for removing endotoxin from protein preparations, can solve problems such as erroneous results, unsafe use of therapeutic products, confusing research results, etc.

Inactive Publication Date: 2015-12-02
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

They are ubiquitous contaminants in biologics that pose serious problems because they are broad-spectrum cytotoxins that c

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1. Reduction of endotoxin from IgG-endotoxin mixture by combination of allantoin and space exclusion chromatography. Endotoxin was added to 1 mg / mL human IgG (clone her2) in 20 mM Hepes, 150 mM NaCl, pH 7.5 to 3,300 EU / ml. 30% (w / v) allantoin was added to an aliquot of this mixture and allowed to mix for 15 minutes at room temperature. The supernatant was clarified by centrifugation. The protein recovery rate was greater than 50%, and the endotoxin content of the supernatant was reduced by more than 99%, reaching less than 20 EU / ml. 1 mL of the IgG-containing supernatant was then applied to an 8.8 mL gravity column filled with positively charged porous particles (UNOsphereQ, Bio-Rad Laboratories) equilibrated with 20 mM Hepes, 150 mM NaCl, pH 7.5. Next, 15 ml of equilibration buffer was applied and 1 ml fractions were collected at the outlet of the column. Eluted fractions were analyzed by UV absorbance at 280 nm and 254 nm and by LAL kinetic chromogenic endo...

Embodiment 2

[0094] Example 2. Endotoxin reduction from IgG-endotoxin mixtures by space exclusion alone. Endotoxin was added to 1 mg / mL human IgG (clone her2) in 20 mM, Hepes 150 mM, NaCl pH 7.5 to 400 EU / ml. The samples were subjected to space exclusion under the same conditions as described in Example 1. About 90% of the IgG eluted in the pooled fractions 4, 5 and 6, and the endotoxin content was reduced by 99.6%.

Embodiment 3

[0095] Example 3. Endotoxin reduction from protein solutions by a combination of allantoin-mediated co-precipitation and space exclusion chromatography. Endotoxin was added to samples of 1 mg / ml bovine serum albumin (BSA) in 20 mM Hepes 350 mM NaCl pH 7.5. Samples were processed as in Example 1 except that the equilibration and elution buffers for positive space exclusion chromatography contained 350 mM NaCl. Allantoin-mediated co-precipitation reduces endotoxin by more than 99.95%, with protein recovery of more than 90%. SEC did not achieve further reduction of endotoxin, but it effectively removed soluble allantoin from protein samples with protein recoveries exceeding 85%.

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PUM

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Abstract

A method includes (i) adding allantoin in a supersaturating amount to a protein preparation including a desired protein and at least one endotoxin as a contaminant, (ii) removing solids after the adding step to provide a sample for further purification by void exclusion chromatography on a packed particle bed of electropositive particles in a column, the packed particle bed having an interparticle volume, (iii) applying a sample volume to the packed particle bed, wherein the electropositive particles support void exclusion chromatography, and wherein the sample volume is not greater than the interparticle volume, and (iv) eluting a purified sample including the desired protein and a reduced amount of the endotoxin. The method is optionally carried out with only the allantoin treatment or only the void exclusion chromatography.

Description

[0001] Statement of relevant application [0002] This application claims priority to US Provisional Application No. 61 / 768,232, filed February 22, 2013, which is hereby incorporated by reference in its entirety. Background technique [0003] Embodiments disclosed herein relate to methods for reducing endotoxin levels in protein preparations, including unpurified, partially purified, and highly purified preparations. Endotoxins are lipopolysaccharides derived from the cell walls of Gram-negative bacteria. They are ubiquitous contaminants in biologics that pose serious problems because they are broad-spectrum cytotoxins that can confound research results, give erroneous results in diagnostic assays, and render desired therapeutic products unsafe use. This makes it important to have effective materials and methods for their removal from the biological agents in which they reside. Many such materials and methods have been developed. Known examples include treatment of prepara...

Claims

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Application Information

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IPC IPC(8): C07K1/14C07K1/16C07K1/18C07K1/36B01D15/36B01D15/08B01D15/34
CPCB01D15/34B01D15/363B01D15/3847C07K1/18C07K1/30C07K1/34C07K1/36
Inventor 彼得·斯坦利·加尼翁文森特·瓦根尼德
Owner AGENCY FOR SCI TECH & RES
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