Affinity purification process capable of removing host cell protein content

A host cell protein, affinity technology, applied in the biological field, can solve the problems of difficult removal of HCP, difficult purification process, and the content of HCP cannot meet the quality control standards, and achieves a wide range of applications and significant effects.

Active Publication Date: 2018-03-13
WUXI BIOLOGICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, some HCPs can interact with the target protein or adsorb non-specifically with the filler matrix, which makes it very difficult to remove these HCPs in the affi

Method used

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  • Affinity purification process capable of removing host cell protein content
  • Affinity purification process capable of removing host cell protein content
  • Affinity purification process capable of removing host cell protein content

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Effect of low pH eluting 2 buffer in Fc fusion protein affinity chromatography process

[0072] Use 50mM Tris-HAc, 150mM NaCl, pH 7.4 as the equilibration buffer for Step 1a, Step 1c, Step 2a, Step 5a, and Step 5c; 50mM NaAc-HAc, pH 5.5, as the wash 3 buffer for Step 2c 0.1M NaOH solution is used as the disinfectant of step 1b and step 5b; 20% ethanol is used as the preservation solution of step 5d; acetic acid-sodium acetate system, buffer solutions of different pH are used as the rinse 2 buffer solution of step 2b respectively ; Loading with Protein A chromatographic column (JSR A3, 3ml), loading capacity is 24mg / mL and 28mg / mL (24mg / mL: pH4.2, 4.3, 4.4; 28.0mg / mL: pH4.0, 4.5, 5.5, 9.0).

[0073] Rinse the column with 2 times (3 times: for a load of 28 mg / mL) column volume of equilibration buffer (step 1a) and then sanitize the column with 3 times the column volume of NaOH (step 1b); use 5 times the column volume Equilibrate the column with equilibration b...

Embodiment 2F

[0078] Example 2 Effect of adding sodium chloride, calcium chloride and arginine to Fc fusion protein in affinity chromatography process

[0079] Use 50mM Tris-HAc, 150mM NaCl, pH 7.4 as the equilibration buffer for Step 1a, Step 1c, Step 2a, Step 5a, and Step 5c; 50mM NaAc-HAc, pH 5.5, as the wash 3 buffer for Step 2c 0.1M NaOH solution is used as the disinfectant of step 1b and step 5b; 20% ethanol is used as the preservation solution of step 5d; 50mM NaAc-HAc, the buffer solution of pH 5.5 is used as the rinse 2 buffer solution of step 2b; other The wash 2 buffer of step 2b in the comparative example also contains 1M sodium chloride, or 0.5M calcium chloride, or 0.25M arginine amino, or 0.5M arginine; ), the loading capacity was 18mg / mL.

[0080] Rinse the column with 2 column volumes of equilibration buffer (step 1a) and then sanitize the column with 3 column volumes of NaOH (step 1b); equilibrate the column with 5 column volumes of equilibration buffer (step 1c ); load ...

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PUM

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Abstract

The invention provides a novel affinity purification process capable of effectively lowering host cell protein (HCP) content. The affinity purification process specifically includes the steps of firstly, processing affinity filler, and performing sample loading; secondly, using a buffer solution with low pH (3.9-5.6) and an elution buffer solution 2 using sodium chloride, calcium chloride and/or arginine hydrochloric acid as additives to elute a chromatographic column; thirdly, using different pH elution buffer solutions to elute a product, and collecting the product. The affinity purificationprocess has the advantages that materials used by the affinity purification process are evident in HCP removing effect, wide in application range and widely applicable to the affinity purification process of antibody protein, and accordingly technical support is provided for the effective control of HCP content in antibody medicine.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an affinity purification process for effectively removing FC fusion protein and monoclonal antibody host cell protein content. Background technique [0002] Host cell protein (HCP) refers to the protein component derived from host cells, and is an impurity that needs to be removed during the preparation of antibody drugs (including antibodies and FC fusion proteins). Since the main component of HCP is protein, even a small dose may cause an allergic reaction in the body. In addition, part of HCP itself has protease activity and remains in antibody drugs, which may lead to degradation of protein drugs and ultimately affect drug efficacy. If there is an interaction between the residual HCP and the antibody drug, it may block the active site of the antibody drug, which will also lead to a decrease in drug efficacy. Therefore, in the preparation process of antibody drugs, the content o...

Claims

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Application Information

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IPC IPC(8): C07K1/22C07K16/00
CPCC07K1/22C07K16/00
Inventor 刘海宽林森珠韩金玉吴桃利沈克强周伟昌陈智胜
Owner WUXI BIOLOGICS CO LTD
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