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Method for separating and removing oligomer in TNFR-Fc fusion protein

A fusion protein and oligomer technology, applied in the field of protein separation and purification, can solve the problems of not particularly large molecular weight difference, a large amount of processing time, expensive materials and sample volume, etc.

Active Publication Date: 2014-04-09
LUNAN PHARMA GROUP CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional method for removing oligomers is molecular sieve chromatography (Size Exclusion Chromatography SEC), but molecular sieve chromatography is the bottleneck of purification and has many disadvantages: First, the separation effect of molecular sieve chromatography is not complete, and for proteins with not particularly large molecular weight differences , the separation effect is not very good; second, compared with other chromatography techniques, molecular sieve chromatography requires highly concentrated samples, and the sample volume can only be between 0.5% and 4% of the column volume. The column must be thin and long to get good results. The separation effect is poor, requiring a lot of processing time, requiring a large column volume, expensive materials and low sample loading, which is not conducive to scale-up production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Method for separating and removing oligomers in TNFR-Fc fusion protein

[0034] First, protein A affinity chromatography

[0035] Use rProtein A Sepharose FF filler, pack LC50 / 600 plexiglass chromatography column according to the instructions, the gel volume is 500ml, and the adsorption capacity is calculated as 25mg / ml. Wash the chromatography column with distilled water at a flow rate of 30ml / min, and the amount of distilled water is 3 column volumes; pre-equilibrate with a concentration of 0.1mol / L Tris-HCl buffer (pH 7.0), the flow rate is 30ml / min, and the amount of buffer for 3 column volumes.

[0036] First, the protein A affinity chromatography column is equilibrated with 0.02 mol / L Tris buffer (containing 0.2 mol / L sodium chloride, pH 7.0), and the amount of buffer is 2 column volumes;

[0037] Secondly, the supernatant containing the TNFR-Fc fusion protein with a content of 75.0% was adjusted to a pH of 7.0 with a concentration of 3mol / LTris solution, and th...

Embodiment 2

[0051] Method for separating and removing oligomers in TNFR-Fc fusion protein

[0052] First, protein A affinity chromatography

[0053] This step is the same as the first step in Example 1.

[0054] Second, hydrophobic interaction chromatography using Butyl Sepharose 4fast flow as the hydrophobic chromatography medium

[0055] Use Butyl Sepharose 4 fast flow hydrophobic chromatography medium as the hydrophobic interaction chromatography filler to purify the target protein, and pack the BPG100 / 500 chromatography column according to the instructions.

[0056] First, the hydrophobic interaction chromatography column is equilibrated with 0.02 mol / L Tris-HCl buffer solution (containing 1.0 mol / L sulfuric acid at a pH of 7.5), and the amount of the buffer solution is 2 column volumes;

[0057] Then, the TNFR-Fc fusion protein collected by protein A affinity chromatography of the present invention is adjusted to pH 7.5 with a concentration of 3mol / L Tris solution, and with a conce...

Embodiment 3

[0062] Method for separating and removing oligomers in TNFR-Fc fusion protein

[0063] First, protein A affinity chromatography

[0064] The packing and pre-equilibration process of the affinity chromatography column is as described in Example 1.

[0065] First, the protein A affinity chromatography column was equilibrated with 0.1mol / L Tris buffer (containing 0.5mol / L sodium chloride, pH 8.0) with a concentration of 0.1mol / L Tris buffer, and the amount of buffer was 10 column volumes;

[0066] Secondly, the supernatant containing TNFR-Fc fusion protein with a content of 75.4% was adjusted to a pH of 8.0 with a concentration of 3mol / LTris solution, and the sample was loaded at a flow rate of 20ml / min until all the supernatant flowed through the affinity The chromatographic column is then washed with a concentration of 0.1mol / L Tris washing solution (containing a concentration of 0.5mol / L sodium chloride, a concentration of 3.0mol / L urea, and a pH of 8.0), and the amount of bu...

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Abstract

The invention discloses a method for purifying TNFR-Fc fusion protein. The TNFR-Fc fusion protein can be purified via immunoaffinity chromatography and hydrophobic interaction chromatography, that is, the content of oligomers in the protein can be reduced via hydrophobic interaction chromatography after affinity chromatography so as to meet quality requirements of SFDA (State Food and Drug Administration) for biological products. The oligomers in the TNFR-Fc fusion protein can be effectively separated and removed.

Description

technical field [0001] The invention relates to protein separation and purification, in particular to a method for separating and removing oligomers in TNFR-Fc fusion protein. Background technique [0002] TNFR-Fc fusion protein (recombinant human tumor necrosis factor receptor-Fc fusion protein) is a protein polypeptide formed by using genetic engineering technology to recombine human tumor necrosis factor receptor and immunoglobulin Fc segment, that is, Etanercept, Chinese name Yisaipu. It can prevent joint deformation, treat moderate and severe rheumatoid arthritis, and also treat ankylosing spondylitis and severe psoriasis. By specifically binding to tumor necrosis factor (TNF) in the human body, it negatively regulates the activity of TNF, thereby inhibiting the inflammatory response in human bone and joints. Chinese patent application publication CN1417334A discloses the construction and preparation of the recombinant gene of the soluble part of the tumor necrosis fa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K1/22C07K1/20
Inventor 赵志全赵丽丽隋华芹
Owner LUNAN PHARMA GROUP CORPORATION
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