Quality stabilization technique of antitumor protein medicines

A protein-based technology with stable quality, applied in anti-tumor drugs, peptide/protein components, drug combinations, etc., can solve difficult and difficult to achieve high biological activity status and other problems

Inactive Publication Date: 2012-03-07
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] In addition, since the anti-tumor protein drug of the present invention is a survivin (Survivin) mutant containing the carrier system TATm and TAT33m, and since the carrier system TATm and TAT33m are used to drive the Survivin mutant into the cell, if the final obtained If the protein is fully refolded, it may fold the TAT end that mediates the target protein into the cell into the interior of the entire protein, and cannot play its due role. However, if all are denatured, it may make the Survivin mutant It is difficult to enter into the cell to achieve a working state, so it is difficult to apply the usual renaturation or denaturation method to achieve the expected, and it is also difficult to achieve a high biological activity state

Method used

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  • Quality stabilization technique of antitumor protein medicines
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  • Quality stabilization technique of antitumor protein medicines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Quality stabilization process for anti-tumor protein drugs.

[0054] Step 1: Escherichia coli expression and fermentation preparation of the target protein.

[0055] The selected expression system, BL21(DE3) / pET22b+, contains a stringent T7lac promoter, which blocks any gene transcription caused by T7 RNA polymerase, which is beneficial to control basic expression and improve cell growth. Utilize 10L fermenter to ferment, ferment liquid 5L, add 0.1-5% (NH 4 ) SO 4 With 0.01-0.5% salt containing divalent metal cations, to promote the stable accumulation of the target product. The wet weight of the bacteria can reach 300-320 grams, and the expression of the target protein can reach 35.5% of the total protein of the bacteria ( figure 1 ) (determined by bandscan5.0).

[0056] Step 2: crushing of Escherichia coli engineered bacteria and recovery of inclusion bodies.

[0057] Use a high-pressure homogenizer to crush the E. coli fermentation broth under a press...

Embodiment 2

[0092] Example 2: Stability test and biological activity test of protein drug lyophilized powder injection.

[0093] Different batches of proteins were prepared with different concentrations of the target protein, different concentrations of freeze-dried excipients and different freeze-drying processes.

[0094] The first batch: take 10ml of the target protein stock solution with a concentration of 10mg / ml, dilute it to 2mg / ml with protein dilution buffer (control pH 6.5); prepare 2g trehalose with protein dilution buffer to make a 10% solution. Mix the above two solutions in a certain ratio and measure the pH. The packaged products are put into a freeze dryer for freeze drying (the freeze drying curve is as follows: Figure 13 As shown), pre-freeze for 8 hours after entering the box, the temperature of the partition reaches -50°C, the product temperature is -40°C, the vacuum pump is turned on and the product begins to sublimate, and the product temperature is controlled at -...

Embodiment 3

[0102] Example 3: Anti-tumor protein activity detection standards and determination methods.

[0103] Take human breast cancer cell line B-Cap-37 or human pancreatic cancer cell line SW1990 sensitive to this protein as an example, use doxorubicin or paclitaxel as positive drugs, and use the conventional MTT method to add samples to a 96-well plate as follows And the measurement method, due to the consideration of the edge effect, no sample is added on the periphery. Add 20ul of MTT solution (5mg / ml in PBS) to each well of the assay well. Continue to incubate for 4 hours, terminate the culture, and carefully aspirate and discard the culture supernatant in the well. Add 150ul DMSO to each well, shake fully on the shaker for 10min to dissolve the crystals. Set the λ=490nm enzyme-linked detector to measure the absorbance value of each well, and perform statistical processing on the data to calculate the concentration of the target protein drug required to kill 50% of the cells. ...

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Abstract

The invention discloses a quality stabilization technique of antitumor protein medicines, which comprises the following steps: (1) expressing target proteins with colibacillus, and preparing the target proteins by fermentation; (2) crushing the colibacillus engineering bacteria, and recycling the inclusion body; (3) washing the inclusion body, and recycling the target proteins; (4) purifying the target proteins by strong cation column sepharose Fast Flow chromatography; (5) purifying by weak anion column DEAE (diethylaminoethanol) sepharose chromatography, and recycling the target proteins; (6) stably storing the target proteins; and (7) preparing the target protein freeze-dried powder injection by a freeze-drying technique. The target proteins are a type of antitumor-activity survivin mutant proteins supported by TAT (transactivator of transcription) and mutants thereof. The invention simultaneously gives attention to yield, biological activity and stability, and well implements the overall control technique of stable quality of a type of antitumor protein medicines.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, more specifically, to a quality stabilization technology for anti-tumor protein drugs, and a combined control process thereof. Background technique [0002] Tumor is the first major disease that endangers human health, and the use of biological anti-tumor technology has always been the goal pursued by human beings. Anti-tumor protein drugs are the main content and an important part of biological anti-tumor. After years of unremitting research on anti-tumor protein drugs, it has been found that the main stumbling block in the research and development of anti-tumor protein drugs comes from many aspects, among which its production quality and A stable process control process is a very important aspect, which determines to a certain extent whether the anti-tumor protein drug can be developed into clinical application, and also determines the feasibility and economic cost of the large-scale preparati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/17A61K9/19A61K47/48C12P21/00A61P35/00
Inventor 马兴元朱玲石磊唐莉莉郑文云莫剑
Owner EAST CHINA UNIV OF SCI & TECH
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