A kind of purification method of antibody acidic peak

A purification method, antibody technology, applied in the preparation method of peptides, chemical instruments and methods, anti-animal/human immunoglobulin, etc., can solve the problems of large particle size, high cost, short service life, etc., and achieve product recovery High rate, good effect

Inactive Publication Date: 2019-08-16
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cooperating with high-resolution ion exchange filler SP Sepharose High Performance of GE Company, the effect is not satisfactory, and the cost is very expensive. Protein A of GE Company usually needs more than 100,000 liters and has a short service life. In addition, the affinity filler contains Protein A component. After use, protein A will fall off and introduce new impurities. It is necessary to establish a method for detecting protein A and a removal process
Using composite packing and using conventional removal methods, due to the full loading of samples, the particle size is large, the resolution itself is not high, and the effect of removing acidic peaks is not ideal, which puts pressure on the removal of downstream impurities.
[0004] In addition, the combination of other fillers, such as the use of cation exchange and anion exchange, is not very effective in removing the acidic peaks of antibodies in conventional methods.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Composite packing chromatography

[0024] a. Preparation: Feed solution (antibody concentration: 2.18mg / ml) 400ml, use GE's chromatography system AKTA purifer, choose Pall LRC15X080-200V01 column, containing 25ml MEP HyperCel filler. Samples and columns were kept at 4°C throughout.

[0025] b. Equilibration: The equilibration buffer is 150mmol / LPBS (pH7.3), and the flow rate is kept at 4.5ml / min.

[0026] c. Sample loading: The loading volume of each ml filler is 35mg of antibody, and the loading volume is about 400ml.

[0027] d. Washing: use 150mmol / LPBS (pH7.3) equilibration buffer to wash until the baseline is stable.

[0028] 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, use elution buffer II: 50mmol / L citrate + 10mmol / L sodium phenylbutyrate (pH7.0) for elution, separate a tall peak, collect the se...

Embodiment 2

[0039] Composite packing chromatography

[0040] a. Preparation: Feed solution (antibody concentration: 2.5 mg / ml) was 350 ml, using the chromatography system AKTA purifer of GE Company, using Pall LRC15X080-200V01 column, containing 25 ml of MEP HyperCel filler. Samples and columns were kept at 4°C throughout.

[0041] b. Equilibration: The equilibration buffer is 150mmol / LPBS (pH7.3), and the flow rate is kept at 4.2ml / min.

[0042] c. Loading: 35mg of antibody per milliliter of filler, and the loading volume is about 350ml.

[0043] d. Washing: use 150mmol / LPBS (pH7.3) equilibration buffer to wash until the baseline is stable.

[0044]e. Elution: elution in two steps, the first elution buffer is: 30mmol / L citrate + 8mmol / L sodium phenylbutyrate (pH6.5), the eluted peak is the acidic peak and the impurities are discarded. After equilibration, use elution buffer II: 50mmol / L citrate + 8mmol / L sodium phenylbutyrate (pH7.0) for elution, a tall peak is separated, and the seco...

Embodiment 3

[0055] Composite packing chromatography

[0056] a. Preparation: Feed liquid 380ml, antibody concentration 2.24mg / ml, use GE's chromatography system AKTA purifer, choose Pall LRC15X080-200V01 column, containing 25ml MEP filler. Samples and columns were kept at 4°C throughout.

[0057] b. Equilibration: The equilibration buffer is 150mmol / LPBS (pH7.3), and the flow rate is kept at 4.5ml / min.

[0058] c. Loading: The amount of sample loaded per milliliter of filler is 34 mg of antibody, and the sample volume is about 380 ml.

[0059] d. Washing: use 150mmol / LPBS (pH7.3) equilibration buffer to wash until the baseline is stable.

[0060] e. Elution: elution in two steps, the first elution buffer is: 30mmol / L citrate + 5mmol / L sodium phenylbutyrate (pH6.5), the eluted peak is the acidic peak and the impurities are discarded. After equilibration, use elution buffer II: 50mmol / L citrate + 5mmol / L sodium phenylbutyrate (pH7.0) for elution, a tall peak is separated, and the second ...

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PUM

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Abstract

The invention relates to a method for purifying acidic peaks of antibodies. The method implemented at the temperature of 2-8 DEG C particularly includes steps of (1), eluting the acidic peaks by the aid of MEP HyperCel packing and eluent and collecting target peak eluent; (2), carrying out weak cation chromatography on the target peak eluent obtained at the step (1) and eluting the target peaks by the aid of butyrate which is used as buffer salt. The electric conductivity of the eluent for eluting the acidic peaks at the step (1) changes from being low to being high, and the eluent comprises 5-10mmol / L sodium phenylbutyrate. The method has the advantages that removal of the acidic peaks of the antibodies can be reinforced after the acidic peaks are purified by the aid of the method, and the acidic peaks can be within the ranges of 11%-14% under the effective control.

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] During the expression process of monoclonal antibodies, after a variety of translational modifications, it will lead to charge heterogeneity, which is reflected in different isoelectric points, and a series of antibodies with different isoelectric points will be produced. After high-resolution ion-exchange column analysis, continuous peaks appear instead of single peaks. Charge heterogeneity affects the stability and biological activity of monoclonal antibodies. Therefore, in production, charge heterogeneity becomes a key quality attribute and needs to be strictly controlled. [0003] Due to the very small difference in charge heterogeneity, it is difficult to reduce the acidic peak of the antibody to an acceptable range by one step of chromatography. It requires extremely high-resolution ion-e...

Claims

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Application Information

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Patent Type & Authority Patents(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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