Application of sodium phenylbutyrate in purification of antibody acidic peak

A sodium phenylbutyrate, antibody technology, applied in the direction of anti-animal/human immunoglobulin, organic chemistry, peptide preparation, etc., can solve the problems of prolonging the loading time, reducing the recovery rate, co-precipitation, etc. Purification time, reduced operation steps, high recovery effect

Inactive Publication Date: 2016-07-20
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if cationic filler is used, the conductivity and pH of the sample need to be adjusted, which will inevitably dilute the sample and prolong the sample loading time.
In addition, adjusting the pH will generally cause coprecipitation and reduce the recovery rate.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] a. Preparation: take 400ml of material solution (antibody concentration is 2.32mg / ml), use the chromatography system AKTApurifer of GE Company, select PallLRC15X080-200V01 column, and contain 30ml of MEP-HyperCel filler. Samples and columns were kept at 4°C throughout.

[0026] b. Equilibration: The equilibration buffer is 150mmol / LPBS (pH7.3), and the flow rate is kept at 4.5ml / min.

[0027] c. Loading: the amount of sample loaded per milliliter of filler is 29.4 mg of antibody, and the sample volume is 380 ml.

[0028] d. Washing: use 150mmol / LPBS (pH7.3) equilibration buffer to wash until the baseline is stable.

[0029] e. Elution: elution in two steps, the first elution buffer is: 30mmol / L citrate + 10mmol / L sodium phenylbutyrate (pH6.5), the eluted peak is the acidic peak and the impurities are discarded. After equilibration, elute with elution buffer II: 50mmol / L citrate+10mmol / L sodium phenylbutyrate (pH7.0), collect the target peak, and obtain about 40ml.

...

Embodiment 2

[0033] a. Preparation: Take 400ml of the feed solution (antibody concentration is 2.41mg / ml) and add 2 times the volume of water to dilute, add 0.2M acetic acid to adjust the pH to 7.1, filter, and the volume of the feed solution is 1.2L. The chromatography system AKTApurifer of GE Company was used, and the PallLRC15X080-200V01 column was selected, containing 25ml of SPsepharoseFF filler. Samples and columns were kept at 4°C throughout.

[0034] b. Equilibration: The equilibration buffer is 31mmol / LPBS (pH7.3), and the flow rate is kept at 4ml / min.

[0035] c. Loading: The amount of sample loaded per milliliter of filler is 38.6 mg of antibody, and the sample volume is 1200 ml.

[0036] d. Washing: use 40mmol / LPBS (pH7.3) equilibration buffer to wash until the baseline is stable.

[0037] e. Elution: use elution buffer 40mmol / LPBS+200mmol / LNaCl+10mmol / L sodium phenylbutyrate (pH7.3) to elute, separate two peaks, and collect the first peak.

[0038] Reference group 2 was pre...

Embodiment 3

[0041] a. Preparation: Feed solution (antibody concentration: 2.54mg / ml) 450ml, use GE's chromatography system AKTA purifer, choose PallLRC15X080-200V01 column, containing 30ml MEP-HyperCel filler. The sample and column were kept at 8°C throughout.

[0042] b. Equilibration: The equilibration buffer is 150mmol / LPBS (pH7.3), and the flow rate is kept at 4.5ml / min.

[0043] c. Loading: The amount of sample loaded per milliliter of filler is 38.1 mg of antibody, and the sample volume is 450 ml.

[0044] d. Washing: use 150mmol / LPBS (pH7.3) equilibration buffer to wash until the baseline is stable.

[0045] e. Elution: elution in two steps, the first elution buffer is: 30mmol / L citrate + 5mmol / L sodium phenylbutyrate (pH6.4), the eluted peak is the acidic peak and the impurities are discarded. After equilibration, elute with elution buffer II: 50mmol / L citrate+5mmol / L sodium phenylbutyrate (pH7.2), collect the target peak, and obtain about 43ml.

[0046] Reference group 3 was p...

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PUM

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Abstract

The invention relates to an antibody acidic peak purification method. During purification of the antibody acidic peak, sodium phenylbutyrate is added so that a good effect is achieved in application of purification of the antibody acidic peak. During purification of the TNFaplha antibody, content of acidic peak of the antibody can be reduced obviously by means of elution in two steps through buffer solution which contains sodium phenylbutyrate and has conductivity changing from low to high. Therefore, the method has remarkable practical industrial use.

Description

technical field [0001] The invention relates to the field of antibody purification, in particular to a method for purifying acidic peaks of antibodies. Background technique [0002] The acidic peak is a charge heterogeneity related impurity of the mAb. The difference in charge heterogeneity mainly comes from post-translational modifications, such as deamidation, loss of terminal lysine, etc. Acidic peaks are a ubiquitous phenomenon, and high-resolution cationic packings are generally used for purification. However, if a cationic filler is used, the conductivity and pH of the sample need to be adjusted, which will inevitably dilute the sample and prolong the sample loading time. In addition, adjusting the pH will generally cause co-precipitation and reduce the recovery rate. [0003] MEP-HyperCel is a composite filler of Pall Company, which has two mechanisms of hydrophobicity and ion exchange. The feed solution can be directly loaded without adjusting the conductivity an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K16/24C07K1/18C07K1/16
CPCC07K1/165C07K1/18C07K16/241
Inventor 邬君马旭通林小鹊杨彬孙文正苏彦景
Owner SUNSHINE LAKE PHARM CO LTD
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