Recombinant Protein A protein and preparation method of affinity chromatography medium

A chromatographic medium and protein technology, which is applied in the field of protein engineering, can solve the problems of reduced lifespan, unsatisfactory stability, and destruction of the advanced structure of Protein A, and achieve excellent antibody binding ability, good chemical stability, and improved chemical stability.

Active Publication Date: 2021-08-20
PINGHU YOUPU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conditions of in-situ cleaning (0.5mol/L to 1.0mol/L sodium hydroxide solution treatment) are very harsh for Protein A affinity chromatography media, and the direct result is the destruction of Protein A advanced structur

Method used

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  • Recombinant Protein A protein and preparation method of affinity chromatography medium
  • Recombinant Protein A protein and preparation method of affinity chromatography medium
  • Recombinant Protein A protein and preparation method of affinity chromatography medium

Examples

Experimental program
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Effect test

preparation example Construction

[0158] According to another aspect of the present invention, such as figure 1 As shown, a preparation method of an affinity chromatography medium is provided, the ligand of the affinity chromatography medium is the recombinant Protein A protein, and the preparation method of the affinity chromatography medium comprises the following steps:

[0159] S1, the gene for synthesizing recombinant Protein A protein;

[0160] S2. Using the genes synthesized in S1 to construct expression vectors respectively;

[0161] Wherein, the expression vector in S2 is PET23a.

[0162] S3, cultivate and express and purify to obtain the recombinant Protein A solution of the SEQ ID corresponding to the sequence;

[0163] Wherein, the recombinant Protein A solution obtained by culturing and expressing and purifying the corresponding sequence in the S3 specifically includes the following steps:

[0164] S31, using a single recombinant plasmid to transform into Escherichia coli, and carrying out ferm...

Embodiment 1

[0174] The ability and alkali-resistant stability of the affinity chromatography medium prepared from the recombinant Protein A in Table 1 were evaluated using the method described below. The results are shown in Table 1 and image 3 .

[0175] Table 1 Affinity chromatography media with recombinant Protein A evaluated in column (0.5M NaOH)

[0176]

[0177] (1) Determination of the dynamic load of immunoglobulin G (IgG)

[0178]Pack 2ml of resin into the TRICORN TM 5 100 column, equilibrate the column with 20mM phosphate buffer (pH 7.4), and add 20mM phosphate buffer (pH 7) containing human polyclonal IgG (5mg / mL) at a linear flow rate of 300cm / h, and the dynamic binding capacity (DBC) was calculated by the absorbance monitor from the amount of human polyclonal IgG adsorbed when the concentration of human polyclonal IgG in the eluate broke through (Breakthrough) 10% and the volume of the carrier.

[0179] Breakthrough capacity was determined using the AKTA Explorer 10 s...

Embodiment 2

[0186] Repeat Example 1 with the affinity chromatography medium prepared from recombinant Protein A in Table 2, but wash the chromatographic column with 1.0M NaOH instead of 0.5M NaOH. The results are shown in Table 2 and Figure 4 .

[0187] Table 2 Affinity chromatography media with recombinant Protein A evaluated in column (0.5M NaOH)

[0188]

[0189]

[0190] According to another aspect of the present invention, an affinity chromatography medium is provided, which is prepared by the above-mentioned preparation method of the affinity chromatography medium.

[0191] According to yet another aspect of the present invention, an application of recombinant Protein A protein in the preparation of an affinity chromatography medium is provided.

[0192] According to yet another aspect of the present invention, an application of an affinity chromatography medium in antibody separation and purification is provided.

[0193] In summary, with the help of the above-mentioned t...

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Abstract

The invention discloses a recombinant Protein A protein and a preparation method of an affinity chromatography medium. The protein is a mutant of parent immune globulin-binding protein as defined by SEQ ID No 1 and SEQ ID No 2; and the preparation method of the affinity chromatography medium comprises the following steps: S1, synthesizing genes of the recombinant Protein A protein, S2, respectively constructing expression vectors by using the genes synthesized in the S1, S3, culturing, expressing and purifying to obtain a recombinant Protein A solution with a corresponding sequence of SEQ ID, and S4, preparing the affinity chromatography medium by using the recombinant Protein A. The preparation method has the advantages that the chemical stability of the Protein A in alkali liquor is greatly improved by redesigning an amino acid sequence, so that the Protein A with a specific binding effect on an antibody is provided, the Protein A has good chemical stability under an alkaline condition, the binding load of the recombinant Protein A and affinity chromatography medium is remarkably improved, in-place cleaning under 0.5-1.0 M NaOH can be tolerated, and the IgG binding load is 60-90 mg/ml.

Description

technical field [0001] The invention relates to the technical field of protein engineering, in particular to a method for preparing recombinant Protein A protein and an affinity chromatography medium. Background technique [0002] Antibody drugs have excellent curative effect and safety on diseases, and have gradually become important in the treatment of tumors, cardiovascular diseases, especially autoimmune diseases, and are currently the class of drugs with the highest growth rate among biological drugs . Thanks to the construction of high-expression cell lines, improvement of culture medium, and continuous expansion of reactor scale, monoclonal antibody drug technology has developed rapidly in the past few decades. In the preparation process of monoclonal antibody drugs, the purification process of antibodies is particularly important, and the capture of antibodies plays a key role. The Protein A affinity chromatography medium is currently the most widely used and most ...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K1/22B01J20/281B01J20/30
CPCC07K14/00C07K1/22B01J20/281Y02A50/30
Inventor 张洪
Owner PINGHU YOUPU BIOTECH CO LTD
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