Methods of purification of native or mutant forms of diphtheria toxin

A technology of diphtheria toxin and mutants, which is applied in the field of purifying natural or mutant forms of diphtheria toxin, and can solve problems such as heterogeneity

Inactive Publication Date: 2014-04-16
MERCK & CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Proteolytic cleavage (by host proteases or autocatalytically) also occurs frequently for most toxins, resulting in heterogeneity

Method used

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  • Methods of purification of native or mutant forms of diphtheria toxin
  • Methods of purification of native or mutant forms of diphtheria toxin
  • Methods of purification of native or mutant forms of diphtheria toxin

Examples

Experimental program
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Effect test

preparation example Construction

[0083] Several chromatographic supports can be used in the preparation of HA columns, the most commonly used are type I and type II hydroxyapatite. Type I has a high protein binding capacity and better capacity for acidic proteins. Type II, however, has a lower protein binding capacity, but better resolution of nucleic acids and some proteins. The choice of a particular hydroxyapatite type can be determined by the skilled artisan.

[0084] A variety of hydroxyapatite chromatography resins are commercially available, and any available form of the material may be used in the practice of the present invention. In one embodiment of the invention the hydroxyapatite is in crystalline form. The hydroxyapatites used in the present invention may be those that are agglomerated to form particles and sintered at high temperature to form a stable porous ceramic body.

[0085] The particle size of hydroxyapatite can vary widely, but typical particle sizes range from 1 μm to 1000 μm in di...

Embodiment 1

[0145] Example 1: Purification using hydroxyapatite chromatography

[0146] The suitability of hydroxyapatite chromatography was tested. A standard anion-exchange chromatography step was included prior to hydroxyapatite chromatography to increase protein purity to >90% and reduce endotoxins.

[0147] 200 L of fermentation broth was prepared as described above.

[0148] Cell Recovery and Harvesting

[0149] Recovery and concentration of Pseudomonas fluorescens was achieved using continuous centrifugation. The 200L fermentation batch was first cooled to 2 )) of Q / Σ. This step is run to harvest the cytoplasm while the centrate is directed to waste.

[0150] Temperature was controlled to maintain rotor (<8 °C) temperature during the harvest step. After each discharge, the harvested cell slurry was transferred to buckets.

[0151] Osmotic shock and flocculation

[0152] In this step, the CRM 197 Protein release from the periplasm of Pseudomonas fluorescens was achieved ...

Embodiment 2

[0164] Example 2: Commercial-scale purification using CAPTO-MMC™

[0165] Cell Recovery and Harvesting

[0166] Recovery and concentration of Pseudomonas fluorescens was achieved using continuous centrifugation. The 1300L fermentation batch was first cooled to 2 )) of Q / Σ. This step is run to harvest the cytoplasm while the centrate is directed to waste.

[0167] Temperature was controlled to maintain rotor (<8 °C) temperature during the harvest step. After each discharge, the harvested cell slurry was transferred to buckets.

[0168] Osmotic shock and flocculation

[0169] In this step, the CRM 197 Protein release from the periplasm of Pseudomonas fluorescens was achieved by osmotic shock, and flocculants were added to facilitate clarification. When adding resuspension buffer (50% w / v sucrose, 200 mM Tris, 100 mM EDTA, pH 7.5) to resuspend the harvested cell plasma, set agitation to generate vigorous mixing. The resuspended cells were then osmotically shocked by ad...

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Abstract

The present invention relates to the use of hydroxyapatite chromatography and multimodal chromatography, for purification of diphtheria toxin, or a mutant form thereof, from a mixture, for example, a host cell fermentation mixture containing impurities such as host cell proteins and DNA. This invention further relates to the integration of such a method into a multi-step procedure with other fractionation methods for purification of diphtheria toxin suitable for in vitro and in vivo applications.

Description

[0001] Cross References to Related Applications [0002] Not applicable. technical field [0003] The present invention relates to a method for purifying native or mutant forms of diphtheria toxin using hydroxyapatite chromatography and multimodal chromatography. In certain embodiments, the mutant form of diphtheria toxin is CRM 197 . technical background [0004] Diphtheria toxin is Corynebacterium diphtheriae ( Corynebacterium diphtheriae ) toxin-producing strains synthesize and secrete protein toxins. Diphtheria toxin and its mutant forms have found applications in vaccines (as carrier proteins) and anticancer drugs (as targeted therapies). Formaldehyde-inactivated diphtheria toxin has been used in immunization against Corynebacterium diphtheriae since the 1920s. Conjugate vaccines using mutant forms of diphtheria toxin became widely available in the 1980s. See Shinefield, 2010, Vaccine 28:4335-4339. The ability of diphtheria toxin to stimulate T-cell immunity make...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04
CPCC12N9/1077C07K14/34C07H21/04Y02P20/582
Inventor A.R.格尔克T.斯瓦布P.麦休K.瓦伦特
Owner MERCK & CO INC
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