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Method for purifying fusion protein of recombinant human serum albumin

A human serum albumin and fusion protein technology, applied in the field of biomedical purification, can solve the problems of the adverse effect of fusion protein activity, difficult to remove, difficult to enlarge, etc., and achieves the advantages of large-scale operation, reduction of production cost, and service cycle. long effect

Pending Publication Date: 2022-01-04
BEIJING VDJBIO
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, most of the current purification methods for HSA fusion proteins are relatively complicated, the product purity is not high, the protein yield is low (20%), the host cell protein content is high, the cost is high, and it is not easy to scale up. These are the difficulties faced in the purification of fusion proteins
Especially when the HSA fusion protein secreted by mammalian cells (CHO) is purified, it is found that the fusion protein will produce some degradation products and aggregates in different degrees during the culture process. It is more obvious in culture. Their existence will have adverse effects on the activity of fusion proteins. Because the physical and chemical properties are very similar to fusion proteins, it is difficult to remove them by some conventional chromatography methods, especially host cell proteins are more difficult to remove. great challenge

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  • Method for purifying fusion protein of recombinant human serum albumin
  • Method for purifying fusion protein of recombinant human serum albumin
  • Method for purifying fusion protein of recombinant human serum albumin

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0041] Embodiment 1: the method for the fusion protein of purifying recombinant human serum albumin (HSA) and interleukin 2 (IL-2)

[0042] The method of this embodiment mainly includes the following steps:

[0043] For the HPLC purity determination pattern of the fusion protein of recombinant human serum albumin (HSA) and interleukin 2 (IL-2), see figure 1 .

[0044] 1. Supernatant pretreatment: Obtain the cell supernatant from the upstream, add calcium chloride solution to make the final concentration of the supernatant 10mM, then adjust the pH of the supernatant to 5.0 with 1M phosphoric acid, and let stand at room temperature for 1 hour Left and right, the precipitate and some impurities were removed by deep filtration to obtain the acidified supernatant.

[0045] 2. Use SP HP high-resolution strong cation chromatography column to capture and purify the acidified supernatant obtained in step 1, obtain a large amount of target protein and remove most impurities at the sam...

Embodiment 2

[0096] Embodiment 2: the method for the fusion protein of purifying recombinant human serum albumin (HSA) and interleukin 2 (IL-2)

[0097] The method of this embodiment mainly includes the following steps:

[0098] 1. Supernatant pretreatment: Obtain the cell supernatant from the upstream, add calcium chloride solution to make the final concentration of the supernatant 10mM, then adjust the pH of the supernatant to 5.5 with 1M phosphoric acid, and let stand at room temperature for 1 hour Left and right, the precipitate and some impurities were removed by deep filtration to obtain the acidified supernatant.

[0099] 2. Use SP HP high-resolution strong cation chromatography column to capture and purify the acidified supernatant obtained in step 1, obtain a large amount of target protein and remove some impurities at the same time. The specific operation steps are as follows:

[0100] 2.1 Solution preparation

[0101] Equilibrium buffer: 25mM sodium acetate + 50mM sodium chlor...

experiment example 1

[0150] The removal of impurities is an important content in the development of biotechnology drug purification process, especially the removal of host proteins. There are no clear regulations on host protein residues in the world. For antibody drugs, the usual host protein residue content is 100ng / mg protein. In the process of purification process development, the present invention particularly compares the removal effects of different purification processes on host proteins.

[0151] The purification process of Comparative Example 1 and Comparative Example 2 is different from that of Example 1, and the host protein (HCP) removal effect is obviously different. The results are shown in Table 1.

[0152] Table 1 Different purification processes and their host protein removal effects

[0153] experiment number Purification process protein concentration HCP content Example 1 SP HP→Q HP→Capto Pheny 3.95mg / mL 65ng / mg Comparative example 1 SP HP→Q H...

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Abstract

The invention discloses a method for purifying fusion protein of recombinant human serum albumin. The method comprises the following steps of: finely purifying supernate of the fusion protein through acidification precipitation, cation exchange chromatography, anion exchange chromatography and hydrophobic chromatography in sequence, so that process-related impurities and product-related impurities in the fusion protein are basically removed, a high-quality product conforming to pharmaceutical research can be obtained, and the problems of low protein purity and low yield during large-scale production are effectively solved.

Description

technical field [0001] The invention belongs to the field of biological medicine purification methods, in particular to a method for purifying fusion proteins expressing recombinant human serum albumin (HSA) in mammalian cells. Background technique [0002] Human serum albumin (HSA) is a very important natural protein in blood circulation, accounting for half or more of the total serum protein content. The molecular weight is 66KDa, it cannot be filtered by the glomerulus under normal circumstances, and its half-life in serum is about 3 weeks. It has good internal environment compatibility and low immunogenicity. Studies have shown that using human serum albumin as a carrier, the fusion protein expressed by fusing a therapeutic protein or polypeptide with human serum albumin not only maintains the biological activity of the original protein or polypeptide, but also significantly reduces the clearance of the drug in the body Rate, thereby prolonging the half-life of the drug...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/30C07K1/18C07K1/20C07K1/34C07K1/36C07K1/14
CPCC07K1/30C07K1/18C07K1/20C07K1/34C07K1/36C07K1/14
Inventor 成健伟李绍奎田新生李自强张筠
Owner BEIJING VDJBIO