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Method for purifying antibody or Fc fusion protein

A fusion protein and antibody technology, applied in the field of bioengineering, can solve the problems of low elution pH, interference with IEC-HPLC analysis and detection, low mechanical strength, etc., and achieve the effects of mild elution conditions, reduced detection interference, and easy operation.

Pending Publication Date: 2022-05-06
SUZHOU SUNCADIA BIOPHARM CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are the following problems in sample preparation using the above-mentioned fillers: 1) The dynamic load is sensitive to retention time, which is not suitable for high flow rate operation; 2) The elution pH is usually low, which is likely to cause changes in product properties, resulting in test results that cannot reflect the true nature of the sample quality
In addition, agarose-based packing materials like MabSelect SuRe LX and Praesto Jetted 50 have low mechanical strength of the matrix and a maximum pressure resistance of 3 bar, which is not suitable for high flow rate operation
Generally, for affinity eluted samples, alkali needs to be added to neutralize the pH, which is cumbersome and easily increases the sample conductance, which interferes with IEC-HPLC analysis and detection

Method used

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  • Method for purifying antibody or Fc fusion protein
  • Method for purifying antibody or Fc fusion protein
  • Method for purifying antibody or Fc fusion protein

Examples

Experimental program
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specific Embodiment approach

[0212] Further description below in conjunction with the examples, but the scope of the examples is not limited.

[0213] For the experimental methods that do not specify specific conditions in the examples or test examples, usually follow the conventional conditions, or follow the conditions suggested by the raw material or commodity manufacturers. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Methods in Current Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents without specific sources indicated are conventional reagents purchased in the market.

Embodiment 1

[0215] Use an affinity chromatography column equipped with 1mL UniMab 50 packing material (purchased from Suzhou Nawei Technology Co., Ltd.), and use 20mM phosphate buffer (pH7.0) to equilibrate the affinity chromatography column at a flow rate of 5mL / min for 10 times the column volume , Load 5 mL of cell clarified solution containing anti-PD-1 antibody onto the affinity chromatography column at a flow rate of 5 mL / min. Wash the affinity chromatography column with 20mM phosphate buffer (pH7.0) at a flow rate of 5mL / min for 3 times the column volume, and continue to wash the affinity chromatography column with 20mM phosphate buffer+1M NaCl (pH6.0) at a flow rate of 5mL / min The chromatography column was 3 times the column volume, and 20 mM phosphate buffer (pH 7.0) was used to wash the affinity chromatography column 3 times the column volume at a flow rate of 5 mL / min. Use 20mM citrate buffer (pH4.0) to carry out product elution at a flow rate of 5mL / min and collect the eluted f...

Embodiment 2

[0218] With the affinity chromatography column that 7mL UniMab 50 packing material is housed, adopt 50mM Tris-HAc (pH7.9) to equilibrate affinity chromatography column 6 times column volumes at the flow rate of 7mL / min, 15mL containing the antibody in embodiment 1 The cell clarified solution of PD-1 antibody was loaded onto the affinity chromatography column at a flow rate of 7 mL / min. 50mM Tris-HCl+150mM NaCl (pH7.6) at a flow rate of 7mL / min to wash the affinity chromatography column for 6 times the column volume, and 50mM Tris-HAc+1M NaCl (pH7.6) at a flow rate of 7mL / min to continue Wash the affinity chromatography column for 6 times the column volume, and wash the affinity chromatography column for 6 times the column volume at a flow rate of 7 mL / min with 50 mM Tris-HAc (pH 7.9). 50mM acetate buffer (pH4.3) was used for product elution at a flow rate of 7mL / min and the eluted fractions were collected. Then wash the affinity chromatography column with 1 M acetic acid at a...

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Abstract

The invention provides a purification method of an antibody or an Fc fusion protein. Specifically, the present disclosure provides a method of purifying an antibody or an Fc fusion protein using protein A affinity chromatography using an elution buffer having a pH of about 4.0 to 5.0. The method is particularly suitable for low-pH-sensitive antibodies or Fc fusion proteins, and has the characteristics of simplicity in operation, high speed and mild elution conditions.

Description

technical field [0001] The disclosure belongs to the field of bioengineering, and in particular relates to a method for purifying antibodies or Fc fusion proteins using protein A affinity chromatography. [0002] technical background [0003] Antibody drugs are currently the most promising biotechnological drugs. They have the advantages of good targeting, strong specificity, and low toxic and side effects. They are mainly used for the treatment of malignant tumors and autoimmune diseases. The global monoclonal antibody market continues to increase and has become the focus of competition for biopharmaceuticals in various countries. [0004] In recent years, domestic pharmaceutical companies have also accelerated the deployment of antibody drugs. As biological macromolecules, antibody drugs are technically difficult to develop and have a long development cycle. However, the market demand and fierce competition of antibody drugs force companies to speed up the research and de...

Claims

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Application Information

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IPC IPC(8): C07K16/00C07K19/00C07K1/22
CPCC07K16/00C07K1/22C07K2319/30
Inventor 童红飞王宏伟陈炼
Owner SUZHOU SUNCADIA BIOPHARM CO LTD