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A magnetic bead method for extracting residual dna from recombinant protein products

A recombinant protein, magnetic bead method, applied in the field of molecular biology, can solve the problem of low DNA recovery rate

Active Publication Date: 2016-08-24
GENOR BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The technical problem to be solved by the present invention: the problem of low DNA recovery rate when using the existing magnetic bead method to extract DNA in recombinant protein products

Method used

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  • A magnetic bead method for extracting residual dna from recombinant protein products
  • A magnetic bead method for extracting residual dna from recombinant protein products
  • A magnetic bead method for extracting residual dna from recombinant protein products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Using conventional conditions to extract the DNA in blank buffer, BSA (bovine serum albumin) solution and antibody 1 stock solution

[0032] The blank buffer (DDB), BSA solution (44mg / ml) and antibody 1 stock solution (44mg / ml) were used as samples, and CHO DNA was added to them, DNA extraction and PCR quantification were performed using conventional methods, and the DNA recovery rate was calculated. Taking a single sample as an example, the steps are described as follows:

[0033] 1) Sample treatment: take nine 2ml DNA low-adsorption centrifuge tubes, add 100μl sample each, adjust the NaCl concentration to about 0.5M, and adjust the pH to 7-8, add 4pg CHO DNA to 3 sample tubes, and 3 samples 40 pg of CHO DNA was added to the tube.

[0034] 2) Digestion: Add 70 μl of proteinase K solution (3 mg / ml, ie 1×PK) to the sample tube, and incubate at 56° C. for 30 minutes.

[0035] 3) DNA binding: After enzyme digestion, add 360 μl freshly prepared lysate, 30 μl mag...

Embodiment 2

[0040] Example 2 Optimizing Enzyme Cutting Conditions

[0041] Taking the stock solution of Antibody 1 (44mg / ml) as the sample, the cleaning steps and DNA elution conditions of the conventional method were improved, and on this basis, the effects of different digestion conditions on the DNA recovery rate were compared. Proceed as follows:

[0042] 1) Sample treatment and enzyme digestion: See Table 1 for sample treatment and enzyme digestion conditions.

[0043] A. Take 100μl sample in a 2ml DNA low-adsorption centrifuge tube, adjust the NaCl concentration to about 0.5M, adjust the pH to 7-8, record it as TS, and add 3.9pg CHO DNA to each 100μl sample above, as a recovery rate sample, Denoted as TS-Sp. Three replicates were made each, and TS and TS-Sp samples were used for experiments No. 1-6.

[0044] B. First dilute the sample 4 times with PBS, take 100 μl of the sample diluent in a 2ml DNA low-adsorption centrifuge tube, adjust the NaCl concentration to about 0.5M, adjus...

Embodiment 3

[0053] Embodiment 3 optimizes elution time

[0054] Taking the stock solution of Antibody 1 (44mg / ml) as the sample, the cleaning steps and enzyme digestion conditions of the conventional method were improved, and on this basis, the effects of different elution times on the DNA recovery rate were compared. Proceed as follows:

[0055] 1) Sample preparation: first dilute the sample 4 times with PBS, take 100 μl of the sample diluent in a 2ml DNA low-adsorption centrifuge tube, adjust the NaCl concentration to about 0.5M, adjust the pH to 7-8, and record it as PC. Due to the dilution of the initial sample, the amount of DNA added in the sample used for the sample recovery rate should be reduced accordingly, and 0.9 pg of CHO DNA was added to every 100 μl of the sample dilution, which was recorded as PC-Sp0.9. Each of PC and PC-Sp0.9 did 6 repetitions.

[0056] 2) Digestion: add 70 μl proteinase K solution to the sample tube), and incubate overnight at 37°C.

[0057] 3) DNA bi...

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Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a paramagnetic particle method for extracting residual DNAs (Deoxyribose Nucleic Acids) from a recombinant protein product. The invention discloses a paramagnetic particle method for extracting residual DNAs (Deoxyribose Nucleic Acids) from a recombinant protein product. The method comprises a sample preparing step, an enzyme digestion step, a DNA combining step, a DNA cleaning step and a DNA eluting step, and is characterized in that in the enzyme digestion step, the mass ratio of protease K to proteins in the recombinant protein product is at least 1:6, and incubation is performed at the temperature of 37 DEG C for not less than 10 hours; and in the DNA eluting step, a water bath is performed at the temperature of 70 DEG C for not less than 10 minutes. Compared with the conventional method, the method has the advantages that the DNA recovery rate can be raised to over 50 percent, the recovery rate of a trace quantity of DNAs (less than 1pg) can also exceed 70 percent and can be stabilized between 70 percent and 130 percent, and relevant statutable requirements are met. The method is suitable for extracting residual DNAs from an intermediate and a stock solution in the preparation process of the recombinant protein product, can be used for monitoring the extraneous DNA removing capability of a purifying process, and meets the batch inspection requirement of residual DNAs in the stock solution; and an effective method is provided for the extraction of residual DNAs from the recombinant protein product.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a magnetic bead method for extracting residual DNA from recombinant protein products. Background technique [0002] With the development of biomedical technology, more and more mammalian cells are used to produce recombinant proteins, such as: monoclonal antibodies, cytokines, hormones, growth factors, and other enzymes, coagulation factors, etc. The treatment areas involved are becoming more and more extensive, including endocrinology, cardiovascular and cerebrovascular, surgery, blood diseases, anti-tumor and so on. The amount of recombinant protein products has increased from micrograms to milligrams or even grams as the demand for clinical therapeutic effects has increased. There are also more and more biological products that require long-term repeated medication. This makes our technical review department have to pay more attention to the safety conc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 朱红枚王少雄王洪芳吕品
Owner GENOR BIOPHARMA
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