Methods for reducing aggregate content of protein preparations by treatment with aryl anions

A protein and anion technology, which is applied to the materials of anion exchangers, organic anion exchangers, inorganic anion exchangers, etc., and can solve the problems that reagents have not been used yet

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

This reagent has not been used in the field of protein fractionation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1. Experiments were performed to determine the dynamic range of methylene blue at pH 5.2 and physiological conductivity. The sample was a cell culture harvest containing approximately 1 g / L IgG antibody, 310,010 ppm host protein, and 10.5% aggregates. In separate experiments, methylene blue was added to final amounts of 0.01%, 0.05%, 0.1% and 0.5%. The mixture was incubated for 2 hours and samples were withdrawn for analysis. Antibody recoveries were 106%, 62%, 21% and 0%. Host protein content was 239,175 ppm, 246,497 ppm, 714,356 ppm, 0 ppm. Aggregate contents were 8.6%, 4.4%, 14.6%, 21.8%. Increases in host proteins and aggregates at higher methylene blue concentrations were judged to reflect loss of IgG.

Embodiment 2

[0063] Example 2. The 4 treated samples from Example 1 were further treated by exposure to electropositive metal affinity particles in the form of TREN40high (Bio-Works) in an amount of 5% v / v. The recoveries of IgG after two steps (Example 1 treatment + Example 2 treatment) were 90%, 91%, 87%, and 0%. Aggregates were reduced to less than 1%, less than 2%, less than 2% and not measurable (not detected). Host proteins were reduced to about 140,000 ppm, about 177,000 ppm, about 60,000 ppm and non-measurable amounts (not detected). These results were unexpected because the recovery values ​​mostly increased due to the particle contacting step, and because they revealed a surprising synergistic effect.

Embodiment 3

[0064] Example 3. IgG-containing cell cultures were treated by adding allantoin to a concentration of 1%, then lowering the pH to 5.2 and adding methylene blue to a final amount of 0.1%. The mixture was incubated for 2 hours, then TREN particles were added to a final amount of 5% v / v as described in Example 3, and mixed and incubated for an additional 4 hours. Solids were removed by centrifugation, the pH was titrated to 7, and the liquid was passed through a PC1 positive depth filter (Sartorius). IgG recovery was 84%. Aggregates were reduced from an initial 13.4% to 0.4%. Host protein decreased from an initial 234,557ppm to 47,746ppm.

[0065] Those of ordinary skill in the art will understand how to scale up or scale down experimental results such as those described in the above examples to whatever volume is necessary to meet their particular requirements.

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Abstract

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

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 a result of chromatin depletion (Gan et al., J. Chromatography A 1291 (2013) 33-40). Ethacridine has a long history as a protein precipitant. [0003] The aryl anionic dye methylene blue is used for tissue staining and has been used to mediate electron transfer in microbial fuel cells. Methylene blue is also known by t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/18B01D15/36
CPCC07K1/32B01D15/327B01D15/36B01D15/363B01D15/3828C07K1/145C07K1/34C07K1/36C07K16/00
Inventor 彼得·斯坦利·加尼翁
Owner AGENCY FOR SCI TECH & RES
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