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Method for removing nucleic acid from protein products

A protein product and nucleic acid technology, which is applied in the preparation methods of peptides, immunoglobulins, chemical instruments and methods, etc., can solve problems such as difficulty in meeting the needs of antibody drugs, and achieve good clinical application prospects, simple processing methods, and safety. Good results

Active Publication Date: 2015-06-10
信立泰(苏州)药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, general-purpose sub-exchange chromatography can remove a large amount of nucleic acid substances, but the residual amount of nucleic acid in the finished product after treatment is often greater than 1 pg / mg protein, sometimes even as high as tens of pg / mg protein, which is difficult to meet the needs of antibody drugs

Method used

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  • Method for removing nucleic acid from protein products

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1 Nucleic acid removal method of the present invention

[0020] 1. Test material

[0021] Antibody protein sample acquisition:

[0022] Step 1. Preparation of a CHO cell capable of expressing an anti-CD20 monoclonal antibody:

[0023] Preparation of CHO cells expressing anti-CD20 monoclonal antibody: The dhfr (dihydrofolate reductase) expression unit in the pSV2-dhfr vector (ATCC product) was cloned into the pCDNA3.1 (+) vector (Invitrogen Company product), the mammalian cell expression vector pBF01 capable of expressing DHFR was constructed.

[0024] According to literature reports (US Patent, US6399061), the light chain and heavy chain gene fragments of the recombinant anti-CD20 monoclonal antibody were synthesized by chemical synthesis technology; the synthetic gene fragments were respectively cloned into pBF01 vector by DNA recombination technology, and express The recombinant expression vectors pBF01-CD20L and pBF01-CD20H of the light chain and heavy ...

Embodiment 2

[0055] Embodiment 2 Nucleic acid removal method of the present invention

[0056] 1. Test material

[0057] With embodiment 1.

[0058] 2. Nucleic acid removal method of the present invention

[0059] Except that the balance solution was changed to the balance solution in Table 1 below, the other conditions were the same as in Example 1.

[0060] Table 1 Changes in Balance Solution

[0061]

[0062] 3. Detection and analysis

[0063]With embodiment 1.

[0064] 4. Experimental results

[0065] Table 2 Test results

[0066] Numbering

Original nucleic acid content

Nucleic acid content after purification

Antibody yield or purity of the sample after the method of the present invention is processed

1

8.5pg / mg protein

Less than 0.1pg / mg protein

Protein purity 97%, yield 98%

2

2.7pg / mg protein

Less than 0.1pg / mg protein

Protein purity 96%, yield 99%

3

3.9pg / mg protein

Less than 0.1pg / mg protein

Pro...

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Abstract

The invention discloses a method for removing nucleic acid from protein products. The method is used for removing nucleic acid by adopting a membrane chromatography method, and comprises the following steps: a, balancing a membrane chromatographic column by using equilibrium liquid; b, regulating the pH value and electrical conductivity of a to-be-treated sample to be in accordance with the equilibrium liquid; c, feeding the sample, and collecting the effluent at 280nm ultraviolet absorption peak; d, washing with the equilibrium liquid, and collecting the effluent at 280nm ultraviolet absorption peak; e, combining the effluent in the step c and the effluent in the step d. The method can be used for effectively removing nucleic acid, and has the advantages of high protein reclaiming rate, simple treatment method and good process stability; and the protein product treated by the method has the nucleic acid content less than 0.1pg / mg protein, and has good safety and excellent clinical application prospects.

Description

technical field [0001] The invention relates to a nucleic acid removal method in protein products. Background technique [0002] Protein drugs produced by genetic engineering technology need to strictly control the residual host nucleic acid (DNA) content, especially the clinical application dose of antibody protein drugs is large, so the nucleic acid content needs to be controlled within a very low limit. [0003] At present, general-purpose sub-exchange chromatography can remove a large amount of nucleic acid substances, but the residual amount of nucleic acid in the finished product after treatment is often greater than 1 pg / mg protein, sometimes even as high as tens of pg / mg protein, which is difficult to meet the needs of antibody drugs. [0004] There is a need to provide a new nucleic acid removal method. Contents of the invention [0005] In order to solve the above problems, the present invention provides a new nucleic acid removal method and a protein product pr...

Claims

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Application Information

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IPC IPC(8): C07K16/00C07K1/36C07K1/34C07K1/16
Inventor 罗天学荣艳珍罗培文
Owner 信立泰(苏州)药业有限公司
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