Methods for reducing chromatin content in protein preparations by treatment with alkyl cations

A protein and alkyl cation technology, applied in separation methods, peptide preparation methods, immunoglobulins, etc., can solve the problems in the field of protein fractionation and separation without application

Inactive Publication Date: 2016-10-26
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is not known to be used in the field of protein fractionation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1. Independent effect of cetyltrimethylammonium bromide (CTAB) relative to ethacridine. Cell culture harvest containing IgG1 monoclonal antibody at a concentration of about 2 g / L was clarified by centrifugation and microfiltration. The aggregate content was 21.2% and the host protein content was 225,906 IgG parts per million. In a control series of experiments, different aliquots of the clarified harvest were mixed with 0.01%, 0.02% and 0.05% ethacridine and incubated with agitation for 2 hours. In another series, CTAB was added at the same concentration to separate aliquots. Solids were removed by centrifugation. CTAB reduced aggregates to 2.5-2.6%. Ethacridine reduced aggregates to 1.8-2.0%. However, it was consistently observed that ethacridine converted the light chain subpopulation into distinct light chain dimers, whereas CTAB did not.

Embodiment 2

[0066] Example 2. CTAB mixed with allantoin followed by contact with a functionalized solid. Cell culture harvest containing IgG1 monoclonal antibody at a concentration of about 2 g / L was clarified by centrifugation and microfiltration. The aggregate content was 10.5% and the host protein content was 277,980 ppm IgG. Allantoin was added in an amount of 1% w / v. CTAB bromide was added in an amount of 0.05% and the mixture was incubated for 2 hours. Reduced aggregates to 2.5% and reduced host protein to 156,002 ppm. Positively charged particles (Bio-Works TREN high) were added in an amount of 5% v / v and incubated with mixing for 4 hours. Solids were removed by centrifugation and microfiltration. Aggregates were reduced to 1.2% and host protein was reduced to 127,959 ppm with an IgG recovery of 92%.

Embodiment 3

[0067] Example 3. In a parallel example to Example 2 and using the same source material, but where 0.05% CTAB was replaced by 0.05% methylene blue, aggregates were reduced to 5.4% compared to CTAB which would aggregate CTAB reduced host protein to 2.5% and reduced host protein to 223,612 ppm, compared to 156,002 ppm for CTAB. Methylene blue reduced aggregates to 1.2% and host protein to 94,780 after exposure to TREN particles.

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Abstract

A method of reducing aggregate content in a preparation having a target protein includes contacting the preparation with an alkyl cation to form a mixture, and contacting the mixture with at least one functionalized solid to remove excess alkyl cation.

Description

Background technique [0001] Embodiments disclosed herein relate to methods for enhancing the purification of proteins, including antibodies. They relate in particular to methods for reducing the level of aggregates and can be combined with cell culture harvest clarification methods. They further involve the integration of these capabilities with other purification methods to achieve a desired level of protein purity. [0002] Aggregate removal is an important aspect of protein purification. Low concentrations of the yellow fluorescent heterocyclic dye ethacridine have been shown to reduce the aggregate content of antibody preparations, and this result may be due in part to chromatin depletion (Gan et al., J. Chromatography A 1291 (2013) 33-40 ). Ethacridine has a long history as a protein precipitant. [0003] The surfactant cetrimonium bromide is used as a topical antiseptic. It is also used to create gold nanoparticles and is widely used in hair care products. It is us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/16B01D15/26
CPCB01D15/265B01D15/327B01D15/361B01D15/3828C07K1/145C07K1/32C07K1/16C07K1/18C07K1/36C07K16/00
Inventor 彼得·斯坦利·加尼翁
Owner AGENCY FOR SCI TECH & RES
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