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Method for purifying plasmid DNA

A plasmid, stable technology, applied in the field of plasmid DNA liquid formulations, can solve problems such as degradation reactions affecting DNA stability, increasing the risk of contamination and/or degradation, etc.

Inactive Publication Date: 2007-09-19
申特莱恩公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, long-term storage of plasmid DNA drugs leads to many degradation reactions that affect DNA stability
To overcome these challenges, plasmid DNA is often lyophilized for storage at temperatures extending to room temperature, but then requires additional reprocessing steps, increasing the risk of contamination and / or degradation

Method used

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  • Method for purifying plasmid DNA
  • Method for purifying plasmid DNA
  • Method for purifying plasmid DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0219] The adjustment of the diameter to the rate used is derived from the calculation of the Reynolds number in the coils of the continuous cracking system. Because the following analysis assumes that the fluid properties are Newtonian, the calculations reported below are only fully valid in B1a and to some extent in B2.

[0220] The value of the Reynolds number allows one skilled in the art to determine the type of characteristic encountered. Here, we will only focus on fluid flow in pipes (hydraulic engineering).

[0221] 1) Non-Newtonian fluid

[0222] The two most commonly encountered non-Newtonian fluids in industry are Bingham and Ostwald de Waele.

[0223] In this case, the Reynolds number (Re) is calculated as follows:

[0224] Re N is the generalized Reynolds number

[0225] Re N =(1 / (2 n-3 ))×(n / 3n+1) n ×((ρ×D n ×w 2-n ) / m) (1)

[0226] D: Inner diameter of cross section (m)

[0227] ρ: Volume mass of fluid (kg / m 3 )

[0228] w: space velocity of the f...

Embodiment 2

[0269] We can decompose the CL system into 5 steps. In a specific embodiment, the configuration is as follows:

[0270] 1) Mixing: cells (in solution 1) + solution 2 (M1 + 3m of 6mm tubing). Cells are initially lysed by SDS, there is no risk of fragmentation as long as the DNA is not denatured.

[0271] 2) End of lysis and denaturation of gDNA (13m of 16mm tubing).

[0272] 3) Mixing: lysate + solution 3 (M2 + 3m of 6mm tubing).

[0273] 4) Harvest the neutralized lysate at 4°C

[0274] 5) Settling flocs and large gDNA fragments overnight at 4°C.

[0275] The following conditions can be used to perform sequential lysis:

[0276] - Solution 1: EDTA 10 mM, Glucose (Glc) 9 g / l and Tris HCl 25 mM, pH 7.2.

[0277] - Solution 2: SDS 1% and NaOH 0.2N.

[0278] - Solution 3: acetic acid 2M and potassium acetate 3M.

[0279] - Flow rate 60 l / h: solution 1 and solution 2

[0280] - Flow rate 90 l / h: solution 3.

[0281] - The cells were adjusted to 38.5 g / l with solution 1. ...

Embodiment 3

[0298] The column used was a 1 ml HiTrap column activated with NHS (N-hydroxysuccinimide, Pharmacia), connected to a peristaltic pump (output 2 group, its sequence is as follows:

[0299] 5'-GAGGCTTCTTCTTCTTCTTCTTCTT-3' (SEQ ID NO: 1)

[0300] The buffers used in this example are as follows:

[0301] Coupling buffer: 0.2M NaHCO 3 , 0.5M NaCl, pH 8.3.

[0302] Buffer A: 0.5M ethanolamine, 0.5M NaCl, pH 8.3.

[0303] Buffer B: 0.1M Acetate, 0.5M NaCl, pH 4.

[0304] The column was washed with 6 ml of 1 mM HCl, then the oligonucleotide diluted in coupling buffer (50 nmol in 1 ml) was applied to the column for 30 minutes at room temperature. The column was washed three times with 6 ml buffer A followed by 6 ml buffer B. The oligonucleotides are thus covalently bound to the column via CONH linkages. Store the column in PBS, 0.1% NaN at 4°C 3 , can be used at least 4 times.

[0305] The following two oligonucleotides were synthesized: oligonucleotide 4817: 5'-GATCCGAAGAAGAAG...

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Abstract

The invention relates to plasmid DNA liquid formulations that are stable and stays un-degraded at +4 DEG C to room temperature for long periods of time, and are thus useful for storage of plasmid DNA that are used research, plasmid-based therapy, such as DNA vaccine and gene therapy. The present invention also relates to a method of preserving plasmid DNA in a stable form over time at +4 DEG C to room temperature. The present invention also relates to stable plasmid DNA liquid compositions for use in a method of treatment of the human or animal body by plasmid-based therapy, such as DNA vaccination or gene therapy.

Description

field of invention [0001] The present invention relates to a stable liquid formulation of plasmid DNA, in which the plasmid remains undegraded for a long time at 4°C to room temperature. Thus, the formulations are useful for research or storage of plasmid DNA for use in plasmid-based therapies such as DNA vaccines and gene therapy. Background of the invention [0002] Developments in molecular biology clearly suggest that plasmid-based therapies, specifically in the fields of DNA vaccines and gene therapy, may support effective approaches to disease treatment. A promising way to safely and efficiently deliver normal genes into human cells is through plasmid DNA. Plasmid DNA is covalently closed circular (ccc) or supercoiled bacterial DNA into which a DNA sequence of interest can be inserted. Examples of DNA sequences of interest that can be introduced into mammalian cells include foreign genes, functional or mutated genes, antisense sequences, RNAi or dsRNAi sequences, rib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/06C12N15/10
CPCC12N15/68A61P9/00A61P9/10C12N15/10C12N15/00
Inventor F·布兰彻M·考德N·梅斯特拉利T·吉耶曼D·盖莱
Owner 申特莱恩公司
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