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Method for quantitatively detecting content of intermediate in production process of recombinant human interferon alpha by affinity chromatography,

A technology for quantitative detection of interferon-alpha, applied in the field of biomedicine, can solve problems such as the difficulty in obtaining a large amount of ligand antibodies, and achieve the effects of easy evaluation, improved cognition and control, and simple operation

Pending Publication Date: 2019-02-05
BEIJING TRI PRIME GENE PHARMA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no report on the quantitative analysis of protein intermediates by immunoaffinity chromatography. On the one hand, it is not easy to obtain a large amount of ligand antibodies for specific protein adsorption. On the other hand, the purity of the obtained antibodies must be high to ensure specificity.

Method used

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  • Method for quantitatively detecting content of intermediate in production process of recombinant human interferon alpha by affinity chromatography,
  • Method for quantitatively detecting content of intermediate in production process of recombinant human interferon alpha by affinity chromatography,
  • Method for quantitatively detecting content of intermediate in production process of recombinant human interferon alpha by affinity chromatography,

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Obtaining and affinity analysis of anti-recombinant human interferon alpha monoclonal antibody

[0064] The immune antigen used in the present invention is purified recombinant human interferon alpha, and the immune animals are 6-week-old male BALB / C mice. 40 monoclonal antibody-positive cell lines against recombinant human interferon-alpha were obtained. Among them, BTP-mAb4D9D1E7 is a monoclonal antibody cell line secreting anti-recombinant human alpha interferon and recombinant integrated interferon. The titer of BTP-mAb4D9D1E7 ascites monoclonal antibody is 1:10000-30000. Continuously passaged in vitro for 6 months, the ability to secrete antibodies is stable and specific; the subclass of monoclonal antibodies is IgG 1 .

[0065] The specificity and affinity analysis of the monoclonal antibody to recombinant human alpha interferon and recombinant integrated interferon is as follows:

[0066] Coat 1-10 μg / ml recombinant human alpha interferon and recomb...

Embodiment 2

[0069] Example 2 Method for Quantitative Detection of Recombinant Human Alpha Interferon Alpha 1b Production Process Intermediate Content by Affinity Chromatography

[0070] 1. Construction of engineering bacteria expressing recombinant human alpha interferon alpha 1b

[0071] The gene encoding the recombinant human interferon alpha 1b is inserted into the expression vector pET-23b to obtain a recombinant plasmid, and then the recombinant plasmid is used to transform Escherichia coli BL21 to obtain a recombinant engineered bacterium. The recombinant engineering bacteria were fermented and cultured to induce expression. For details, please refer to CN201010189352.8.

[0072] 2. Ferment engineering bacteria and isolate and purify the target protein expressed in the form of soluble protein or inclusion body

[0073] Protein purification includes crude purification and purification. Ammonium sulfate salt precipitation is used to obtain the crude protein product. The crude produc...

Embodiment 3

[0087] Example 3 Immunoaffinity Chromatography Quantitative Detection of Interferon Process Intermediate Accuracy Verification

[0088] Experimental materials: Instrument: AKTA purifier, chromatographic column: the monoclonal antibody affinity chromatography column of Example 1, mobile phase A: 25mM PBS, pH 7.0, mobile phase B: 0.1M Gly-0.2M NaCl, adjust the pH to 2.5. Loading sample: recombinant human interferon α1b standard substance, the concentration is 5 mg / ml.

[0089] Experimental scheme: flow rate: 1.274cm / min, detection wavelength: 280nm, detection time: 65min, elution condition: 100%A(3CV)-100%B(6CV)-100%A(5CV).

[0090] Experimental procedure: After equilibrating the column with mobile phase A, load 0.2ml, 0.3ml, 0.4ml, and 1ml of interferon standard in sequence, completely collect the elution peak of the target protein, adjust the pH to neutral with NaOH, and detect with the trace lowry method concentration.

[0091] Experimental results: the recovery rate was ca...

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Abstract

The invention relates to a method for quantitatively detecting the content of an intermediate in the production process of recombinant human interferon alpha by affinity chromatography. The method forquantitatively detecting the content of the intermediate in the production process of the recombinant human interferon alpha by affinity chromatography comprises the following steps that (1) engineering bacteria expressing the recombinant human interferon alpha is constructed; and (2) the engineering bacteria is fermented, and the target protein expressed in the form of soluble protein or an inclusion body is separated and purified, wherein the protein purification comprises crude purification and refined purification, a crude protein is obtained by ammonium sulfate salting-out precipitation,the crude protein is purified by DEAE anion exchange column chromatography to obtain the intermediate, the content of the recombinant human interferon alpha in the intermediate is quantitatively detected by a monoclonal antibody affinity chromatography column. The method is suitable for quantitatively detecting the intermediate in the production process of recombinant human interferon alpha and recombinant integrated interferon, and has the advantages of simple, rapid and accurate operation, and can improve the cognition degree and control degree of people on the production process accordingto the obtained interferon concentration data, so that reliable, rapid, direct and easy evaluation of key links of interferon production is achieved so as to realize the purpose of accurate quality control.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for quantitatively detecting the content of a recombinant human alpha interferon process intermediate by affinity chromatography. Background technique [0002] Quality control is an important link in drug R&D and production, a prerequisite for ensuring drug safety and effectiveness, and one of the main topics in new drug research. Protein content determination is one of the most basic analytical methods in the quality control of biological products. At present, the focus of drug content quality control is mainly concentrated on the raw liquid and finished preparations, while ignoring the quality control of the production process and its intermediates. Moreover, due to the complexity of the process of drugs, especially biopharmaceutical products, there will be varying degrees of loss of main component proteins and interference of related proteins in each purification and separ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/558C12N15/21C12N15/70C07K14/56C07K1/36C07K1/30C07K1/18
CPCC07K14/56G01N33/558G01N33/6866
Inventor 杨大军茹莉莉刘娴吴京雷韩志刚程永庆
Owner BEIJING TRI PRIME GENE PHARMA CO LTD
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